AN ABSTRACT OF THE THESIS OF
Melissa M. Sales for the degree of Master of Science in Food Science and Technology
presented on July 25, 2013
Title: An Evaluation of Blackberry Harvest Sanitation and the Ability of Foodborne
Pathogens to Survive in Blackberry Products
Abstract approved:
Mark A. Daeschel
Blackberries, genus Rubus, are an important Oregon agricultural commodity. In
their fresh and processed forms, they offer many health benefits and may be able to
help Americans better achieve fruit consumption recommendations because of
convenience and pleasant sensory qualities. However, the susceptibility of blackberry
products to contamination with bacterial pathogens of human health concern is
unknown. Blackberries have never directly been implicated in a food safety incident;
however, raspberries, also in the Rubus genus, have been the vehicle for hepatitis A,
norovirus, and Cyclospora cayetanensis outbreaks. Furthermore, strawberries were
recently the source of an Escherichia coli O157:H7 outbreak in Oregon.
To better understand the potential for microbial pathogen contamination and
the ability of these microorganisms to survive or grow in blackberry products, several
studies were conducted. Fresh berries from the ‘Obsidian’ and ‘Triple Crown’ cultivars
were evaluated at different harvest periods for the aerobic plate count, coliforms,
yeasts, and molds to establish a baseline microbial population. Environmental samples
were taken from a clean mechanical harvester and then from the same harvester that
had been intentionally left soiled with berry harvest debris to determine the impact of
harvester microbial quality. Samples from ‘Marion’ and ‘Black Diamond’ cultivars were
hand harvested and evaluated for E. coli O157:H7 and Salmonella spp. by rapid
detection methods via the NEOGEN® Reveal® 2.0 systems. Fresh, wild ‘Himalaya’
blackberries and frozen blackberries from the ‘Triple Crown’ cultivar were spot
inoculated with Escherichia coli O157:H7, Salmonella Typhimurium, Listeria
monocytogenes, and Staphylococcus aureus to determine the ability of these
microorganisms to survive on the berry surface. ‘Himalaya’ samples were stored for 3
days at ambient temperatures and ‘Triple Crown’ for 6 months at -23.3°C. Lastly, juice
and wine were made from ‘Marion’ and ‘Black Diamond’ purees. The juices and wines
were used for pathogen survival studies using the aforementioned microorganisms to
better understand what constituents of blackberries may contribute to bactericidal
effects, as well as the survival patterns in these products.
Aerobic plate counts for ‘Obsidian’ and ‘Triple Crown’ cultivars ranged from 3.524.62 log CFU/g of berry with later harvests tending to have higher values. ‘Triple Crown’
mid-late harvest samples were significantly higher than the early harvest samples (p =
0.005). Yeasts and molds ranged from 3.01-4.73 log CFU/g of berry with later harvests
having significantly higher values for both cultivars (p = 0.048 ‘Obsidian’; p <0.001 ‘Triple
Crown’). Coliforms were detected in ‘Obsidian’ mid-harvest and ‘Triple Crown’ earlyharvest samples at 2.10 and 1.40 log CFU/g of berry, respectively. The aerobic plate
counts measured from the clean and intentionally soiled mechanical harvester were not
significantly different. Escherichia coli O157:H7 and Salmonella spp. were not detected
using rapid detection methods in evaluated ‘Marion’ and ‘Black Diamond’ samples.
Escherichia coli O157:H7 was not detectable in fresh or frozen inoculated
samples. Salmonella Typhimurium was detected in 2 frozen samples with 2.95 and 3.21
log reductions. Listeria monocytogenes was only detected in frozen samples and
experienced log reductions ≥ 2.42. Staphylococcus aureus was detectable on every
fresh and frozen berry inoculated with log reductions ranging from 0.67 to 3.48. The
greatest reductions occurred with fresh samples.
Growth of microorganisms was not observed in any juice or wine samples.
Maximum observed survival times in juices ranged from 12 h for L. monocytogenes to
108 h for Salmonella Typhimurium. Maximum survival times in wines were 40 m for
both E. coli O157:H7 and Salmonella Typhimurium, and 80 m for both L. monocytogenes
and S. aureus. Adding ethanol to juice samples to equal that of their counterpart wines
decreased survival time for all microorganisms evaluated by several hours. Increasing
the pH of wines by approximately one unit increased the survival time from minutes to
hours, and in some cases, days.
The overall results suggest that blackberries are not an ideal environment for E.
coli O157:H7, Salmonella Typhimurium, L. monocytogenes, and S. aureus to grow.
However, these microorganisms may be able to survive depending on the type of
blackberry product and its subsequent storage. Many constituents of blackberries may
provide bactericidal activity, with organic acids appearing to have the greatest effect.
©Copyright by Melissa M. Sales
July 25, 2013
All Rights Reserved
An Evaluation of Blackberry Harvest Sanitation and the Ability of Foodborne Pathogens
to Survive in Blackberry Products
by
Melissa M. Sales
A THESIS
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented July 25, 2013
Commencement June 2014
Master of Science thesis of Melissa M. Sales presented on July 25, 2013.
APPROVED:
Major Professor, representing Food Science and Technology
Head of the Department of Food Science and Technology
Dean of the Graduate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature authorizes release of thesis to any reader upon
request.
Melissa M. Sales, Author
ACKNOWLEDGEMENTS
I would like to express my sincerest gratitude to Mark Daeschel, not only for
being an incredibly patient and compassionate mentor, but for also giving me the
freedom and opportunities to discover my strengths. For that, I am truly grateful.
Thank you to David Bryla, Javier Fernandez-Salvador, Renee Harkins, Angela Tseng,
Jooyeoun Jung, George Cavender and Mingyang Lui for all of their time, knowledge, and
assistance with collecting samples. I would like to thank everyone that gave me support
and advice during my time at OSU, especially Dan Smith, Linda Dunn, Lisbeth Goddik,
Joy Waite-Cusic, Brian Yorgey, and Jeff Clawson. Whether you know it or not, you’ve
made many decisions that I’ve had to make a little easier. Thank you to all of the
wonderful and supportive friends that I have made at OSU that I have come to consider
family. I would especially like to thank Jake Mattson for teaching me not to take myself
too seriously and for helping me to become a larger geek than I ever could have become
on my own. I would like to thank my significant other, Zak, for his incredible support and
for never giving up on me during my academic journey even when I was completely
insufferable. I would also like to thank my son, Alex, for being the greatest, most
patient kid I could have ever asked for and for giving me a reason to establish higher
goals. I only wish that my parents, Karen and Gilbert Sales, could have seen me come
this far.
CONTRIBUTION OF AUTHORS
Dr. Joy Waite-Cusic assisted with microbiological data interpretation. Jacob
Mattson assisted with the statistical analysis of harvester and microflora data.
TABLE OF CONTENTS
Page
1. Introduction .................................................................................................................... 1
1.1 Production ................................................................................................................. 1
1.2 Consumption and Health Benefits ............................................................................ 2
1.3 Potential microbial Risks ........................................................................................... 6
1.4 Potential Antimicrobial Properties .......................................................................... 10
2. An Evaluation of Blackberry Harvest Sanitation and the Ability of Escherichia coli
O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus
to Survive on the Surface of Fresh and Frozen Blackberry Fruit ...................................... 13
2.1 Abstract ................................................................................................................... 14
2.2 Introduction............................................................................................................. 16
2.3 Materials and Methods ........................................................................................... 20
2.3.1 Fresh Field Samples .......................................................................................... 20
2.3.2 Direct Pathogen Testing ................................................................................... 21
2.3.3 Mechanical Harvester ....................................................................................... 22
2.3.4 Whole Berry Inoculation................................................................................... 23
2.3.5 Data Analysis..................................................................................................... 25
2.4 Results and Discussion ............................................................................................ 25
2.4.1 Fresh Field Data ................................................................................................ 25
2.4.2 Direct Pathogen Testing ................................................................................... 28
2.4.3 Mechanical Harvester ....................................................................................... 29
2.4.4 Spot Inoculation Studies ................................................................................... 30
2.5 Conclusions.............................................................................................................. 35
3. An Evaluation of the Survival of Escherichia coli O157:H7, Salmonella Typhimurium,
Listeria monocytogenes, and Staphylococcus aureus in ‘Marion’ and ‘Black Diamond’
Blackberry Juice and Wine ................................................................................................ 37
TABLE OF CONTENTS (Continued)
3.1 Abstract ................................................................................................................... 38
3.2 Introduction............................................................................................................. 39
3.3 Materials and Methods ........................................................................................... 42
3.3.1 Juice and Wine Preparation.............................................................................. 42
3.3.2 Juice and Wine Properties ................................................................................ 43
3.3.3 Culture Preparation .......................................................................................... 43
3.3.4 Survival Study Procedure .................................................................................. 44
3.3.5 Data Analysis..................................................................................................... 44
3.4 Results and Discussion ............................................................................................ 44
3.4.1 Properties of Purees, Juices, and Wines........................................................... 44
3.4.2 Survival Study Results ....................................................................................... 45
3.5 Conclusions.............................................................................................................. 55
4. Overall Conclusions and Future Work ......................................................................... 57
Bibliography ...................................................................................................................... 59
APPENDICES ...................................................................................................................... 65
LIST OF FIGURES
Figure
Page
Figure 2.1 Aerobic Plate Count at Various Harvest Times ................................................ 27
Figure 2.2 Yeasts and Molds at Various Harvest Times .................................................... 27
Figure 2.3 Coliforms at Various Harvest Times ................................................................. 28
Figure 2.4 Mechanical Harvester Aerobic Plate Counts ................................................... 29
Figure 3.1 Survival of E. coli O157:H7 in ‘Marion’ Products ............................................. 48
Figure 3.2 Survival of E. coli O157:H7 in ‘Marion’ Wine ................................................... 49
Figure 3.3 Survival of E. coli O157:H7 in ‘Black Diamond’ Products ................................ 49
Figure 3.4 Survival of E. coli O157:H7 in ‘Black Diamond’ Wine ...................................... 50
Figure 3.5 Survival of Salmonella Typhimurium in ‘Marion’ Products ............................ 50
Figure 3.6 Survival of Salmonella Typhimurium in ‘Marion’ Wine ................................... 51
Figure 3.7 Survival of Salmonella Typhimurium in ‘Black Diamond’ Products ................. 51
Figure 3.8 Survival of Salmonella Typhimurium in ‘Black Diamond’ Wine....................... 52
Figure 3.9 Survival of S. aureus in ‘Marion’ Products ....................................................... 53
Figure 3.10 Survival of S. aureus in ‘Marion’ Wine ........................................................... 53
Figure 3.11 Survival of S. aureus in ‘Black Diamond’ Products......................................... 54
Figure 3.12 Survival of S. aureus in ‘Black Diamond’ Wine .............................................. 54
LIST OF TABLES
Table
Page
Table 2.1 Cultivar Information .......................................................................................... 17
Table 2.2 Detection of E. coli O157:H7 and Salmonella spp. in ‘Marion’ and ‘Black
Diamond’ Cultivars ............................................................................................................ 28
Table 2.3 ‘Himalaya’ inoculated with E. coli O157:H7 ...................................................... 30
Table 2.4 ‘Himalaya’ inoculated with Salmonella Typhimurium ...................................... 31
Table 2.5 ‘Himalaya’ inoculated with L. monocytogenes ................................................. 31
Table 2.6 ‘Himalaya’ inoculated with S. aureus ............................................................... 31
Table 2.7 Frozen ‘Triple Crown’ inoculated with E. coli O157:H7 .................................... 32
Table 2.8 Frozen ‘Triple Crown’ inoculated with Salmonella Typhimurium ................... 33
Table 2.9 Frozen ‘Triple Crown’ inoculated with L. monocytogenes ................................ 33
Table 2.10 Frozen ‘Triple Crown’ inoculated with S. aureus ............................................ 34
Table 3.1 Cultivar Information ......................................................................................... 39
Table 3.2 pH and Soluble Solid Content of Blackberry Puree .......................................... 45
Table 3.3 pH, Soluble Solid Content, and Titratable Acidity of Blackberry Juices ............ 45
Table 3.4 pH, Ethanol Content, and Titratable Acidity of Blackberry Wines .................... 45
Table 3.5 Maximum Observed Survival Times of L. monocytogenes in ‘Marion’ and ‘Black
Diamond’ Juices and Wines: All Treatments .................................................................... 52
LIST OF APPENDICES
Appendix
Page
Appendix I. Fresh Field Samples Raw Data, Chapter 2 .................................................... 66
Appendix II. Mechanical Harvester Raw Data and Photos, Chapter 2 ............................ 69
Appendix III. ‘Himalaya’ Raw Data, Chapter 2 ...................................................................... 72
Appendix IV. ‘Triple Crown’ Raw Data, Chapter 2 ................................................................ 74
Appendix V. Juice Raw Data, Chapter 3 ................................................................................ 80
Appendix VI. Juice Variables Raw Data, Chapter 3 ................................................................ 89
Appendix VII. Wine Raw Data, Chapter 3 ............................................................................... 94
Appendix VIII. Wine Variable Raw Data, Chapter 3 .............................................................. 102
Appendix IX. Inocula Raw Data, Chapter 3 .......................................................................... 107
LIST OF APPENDIX FIGURES
Figure
Page
Figure II.1 Harvester Location 1 ........................................................................................ 70
Figure II.2 Harvester Location 2 ...................................................................................... 70
Figure II-3 Harvester Location 3 ...................................................................................... 71
Figure II.4 Harvester Location 4 ...................................................................................... 71
Figure II.5 Harvester Location 5 ........................................................................................ 71
Figure II.6 Harvester Location 6 ........................................................................................ 71
Figure II.7 Harvester Location 7 ........................................................................................ 72
Figure II.8 Harvester Location 8 ........................................................................................ 72
LIST OF APPENDIX TABLES
Table
Page
Table I.1 Aerobic Plate Count Raw Data, Fresh Samples .................................................. 66
Table I.2 Yeasts and Molds Raw Data, Fresh Samples ...................................................... 67
Table I.3 Coliforms Raw Data, Fresh Samples ................................................................... 68
Table II.1 Aerobic Plate Count Raw Data .......................................................................... 69
Table III.1 E. coli O157:H7 on fresh 'Himalaya' Blackberries, Raw Data ....................... 72
Table III.2 Salmonella Typhimurium on fresh 'Himalaya' Blackberries, Raw Data ......... 73
Table III.3 L. monocytogenes on fresh 'Himalaya' Blackberries, Raw Data ...................... 73
Table III.4 S. aureus on fresh 'Himalaya' Blackberries, Raw Data ..................................... 74
Table IV.1 E. coli O157:H7 on Frozen 'Triple Crown' Blackberries, Raw Data ................ 74
Table IV.2 Salmonella Typhimurium on Frozen 'Triple Crown' Blackberries, Raw Data . 76
Table IV.3 L. monocytogenes on Frozen 'Triple Crown' Blackberries, Raw Data ............ 77
Table IV.4 S. aureus on Frozen 'Triple Crown' Blackberries, Raw Data ............................ 78
Table V.1 E. coli O157:H7 in 'Marion' Juice, Raw Data ..................................................... 80
Table V.2 E. coli O157:H7 in 'Black Diamond' Juice, Raw Data ......................................... 81
Table V.3 Salmonella Typhimurium in 'Marion' Juice, Raw Data ..................................... 82
Table V.4 Salmonella Typhimurium in 'Black Diamond' Juice, Raw Data ......................... 83
Table V.5 L. monocytogenes in 'Marion' Juice, Raw Data................................................. 85
Table V.6 L. monocytogenes in 'Black Diamond' Juice, Raw Data .................................... 86
Table V.7 S. aureus in 'Marion' Juice, Raw Data ............................................................... 87
LIST OF APPENDIX TABLES (Continued)
Table
Page
Table V.8 S. aureus in 'Black Diamond' Juice, Raw Data ................................................... 87
Table VI.1 E. coli O157:H7 in 'Marion' Juice with added ethanol, Raw Data.................... 89
Table VI.2 E. coli O157:H7 in 'Black Diamond' Juice with added ethanol, Raw Data ....... 90
Table VI.3 Salmonella Typhimurium in 'Marion' Juice with added ethanol, Raw Data .... 90
Table VI.4 Salmonella Typhimurium in 'Black Diamond' Juice with added ethanol, Raw
Data ................................................................................................................................... 91
Table VI.5 L. monocytogenes in 'Marion' Juice with added ethanol, Raw Data ............... 91
Table VI.6 L. monocytogenes in 'Black Diamond' Juice with added ethanol, Raw Data... 92
Table VI.7 S. aureus in 'Marion' Juice with added ethanol, Raw Data ............................. 92
Table VI.8 S. aureus in 'Black Diamond' Juice with added ethanol, Raw Data ............... 93
Table VII.1 E. coli O157:H7 in 'Marion' Wine, Raw Data ................................................. 94
Table VII.2 E. coli O157:H7 in 'Black Diamond' Wine, Raw Data ...................................... 95
Table VII.3 Salmonella Typhimurium in 'Marion' Wine, Raw Data ................................... 96
Table VII. 4 Salmonella Typhimurium in 'Black Diamond' Wine, Raw Data ..................... 96
Table VII.5 L. monocytogenes in 'Marion' Wine, Raw Data ............................................ 97
Table VII.6 L. monocytogenes in 'Black Diamond' Wine, Raw Data ................................ 98
Table VII.7 S. aureus in 'Marion' Wine, Raw Data ............................................................ 99
Table VII.8 S. aureus in 'Black Diamond' Wine, Raw Data ............................................ 100
Table VIII.1 E. coli O157:H7 in pH adjusted 'Marion' Wine, Raw Data ......................... 102
Table VIII.2 E. coli O157:H7 in pH adjusted 'Black Diamond' Wine, Raw Data ............. 103
LIST OF APPENDIX TABLES (Continued)
Table
Page
Table VIII.3 Salmonella Typhimurium in pH adjusted 'Marion' Wine, Raw Data ........... 103
Table VIII.4 Salmonella Typhimurium in pH adjusted 'Black Diamond' Wine, Raw Data 104
Table VIII.5 L. monocytogenes in pH adjusted 'Marion' Wine, Raw Data ..................... 104
Table VIII.6 L. monocytogenes in pH adjusted 'Black Diamond' Wine, Raw Data ........ 105
Table VIII.7 S. aureus in pH adjusted 'Marion' Wine, Raw Data ................................... 106
Table VIII.8 S. aureus in pH adjusted 'Black Diamond' Wine, Raw Data ....................... 106
Table IX.1 Inocula for Juice Survival Studies ................................................................... 107
Table IX.2 Inocula for Wine Survival Studies .................................................................. 108
An Evaluation of Blackberry Harvest Sanitation and the Ability of Foodborne
Pathogens to Survive in Blackberry Products
1. Introduction
1.1 Production
Blackberries belong to the genus Rubus, subgenus Rubus, and are an important
agricultural commodity in Oregon (Hummer 2010). The United States is the largest
producer of blackberries in the world, with 65% of the United States production
occurring in Oregon in 2005 (Strik and Others 2007). Oregon experienced a 25%
increase in blackberry production between 1995 and 2005, and a 6% increase between
2005 and 2011, a trend that is expected to continue (Strik and Others 2007; USDA
2012).
Oregon is unique in that the majority of cultivated blackberries are trailing types,
which are typically machine harvested. Most states and growing regions predominantly
cultivate erect and semierect cultivars. Trailing types typically and with exception, do
not produce fruit that is firm enough to withstand extensive shipping and handling to be
used for fresh market. Blackberries that are machine harvested are typically destined for
further processing into products such as individually quick frozen (IQF) berries, purees,
jams, jellies, and even wine (Strik and Others 2007). More than 95% of Oregon
blackberries are destined for processing (Strik and Others 2007). In Oregon, the
predominant trailing cultivar is ‘Marion’ and accounted for 51% of harvested
blackberries in 2011 (USDA 2012). ‘Marion’ is known for its desirable sensory qualities
2
for processing (aroma, acid, and sugar profile) (Strik and Others 2007; Du and Others
2010). Another trailing cultivar that has gained popularity in Oregon is ‘Black Diamond’.
‘Black Diamond’ has the added benefit of being thornless and is suitable for processed
products as well as fresh market due to its firmness. This cultivar may be mechanically
or hand-harvested (Strik and Others 2007; Du and Others 2010). The trailing cultivar
‘Obsidian’ is mainly grown for hand-harvested fresh market (Finn and Others 2005).
‘Triple Crown’ is also popular in Oregon and is a semierect cultivar. It is hand-harvested,
which is typical for erect and semierect types (Strik and Others 2007). Trailing types are
harvested between June and August, with erect and semierect types extending into
October, depending on cultivar (Strik and Others 2007; Finn and Strik 2008).
1.2 Consumption and Health Benefits
Consumption of Rubus fruit, stems, and leaves for food and pharmacological
purposes is believed to have begun approximately 10,000 years ago (Hummer 2010). In
Oregon, evidence of Rubus consumption can be dated back to 8000 BCE as determined
by carbon dating of materials found at Newberry Crater in Bend, OR (Hummer 2010).
Ancient Greeks and Romans used nonedible portions of Rubus plants for everything
from hair dye to relieving stomach aches. An interesting record of Hippocrates’ writings
suggest using blackberry leaves soaked in wine to apply to wounds for antiseptic
purposes, a property later understood to be attributed to their tannin content (Hummer
2010). Native populations of North America used Rubus for many ailments related to
3
female reproductive function, including childbirth and menstrual cramps (Hummer
2010). In modern times, blackberries still have much to offer human health.
Obesity is understood to be an epidemic in the United States. Fortunately, rates
of obesity among American adults appear to have reached a plateau, with obese men
comprising 35.5% of the adult population and obese women, 35.8% (Flegal and Others
2012). Obesity is associated with metabolic syndrome which is comprised of a set of risk
factors that increase an individual’s likelihood of developing chronic diseases such as
diabetes and cardiovascular disease (Gropper and Others 2009). One strategy to help
combat obesity in the United States is to increase overall consumption of fruits and
vegetables.
Healthy People is a program under the Department of Health and Human
Services. A primary goal for the Healthy People 2010 objective period was to increase
the consumption of fruits and vegetables among Americans. Specifically, the goal was
to increase the percentage of individuals over the age of 2 years that consume at least 2
servings of fruit a day to 75% and at least 3 servings a day to 50% (CDC 2010). Figures
reported in 2009 showed that the goal was far from being reached with only 32.5% of
adults consuming at least 2 servings of fruit daily, a significant decline from 2000, and
26.3% consuming at least 3 servings of vegetables daily (CDC 2010). Figures for Oregon
were slightly better with 33% consuming at least 2 servings of fruit daily and 30.5%
consuming at least 3 servings of vegetables daily (CDC 2010). To try to improve
consumption of fruits and vegetables in the United States, the Centers for Disease
4
Control and Prevention (CDC) has released a guidance document that outlines a 10 point
strategy to increase consumption. The overall approach to the strategy is to increase
availability and visibility of fruits and vegetables while decreasing cost barriers and
increasing consumer education about where their food is coming from. The CDC feels
that this can be achieved through such practices as increasing farmers markets,
expanding community agriculture programs, and encouraging the availability of fresh
produce in markets that tend to not provide as much choice (CDC 2011).
Blackberries could serve to help increase overall consumption of fruit, especially
when considering the portability and convenience of many blackberry products: for
example, fresh, frozen, and freeze dried fruit and blackberry juice. Blackberries also
have pleasant sensory properties that make them attractive to consumers across age
categories (Du and Others 2010). Nutritionally, they are low in calories, a good source
of fiber, and contain vitamins and minerals (USDA Nutrient Database).
Many components of blackberries could assist with weight management.
Blackberries contain pectin, a soluble fiber. Consumption of pectin has been associated
with increased satiety, which may reduce energy overconsumption (Perrigue and Others
2010). Soluble fiber has also been associated with a reduction in blood lipids, which
reduces the risk for developing cardiovascular disease (Jenkins and Others 2002;
Gropper and Others 2009). Aside from weight loss and management, soluble fiber can
be considered a prebiotic, since it is fermentable in the gut, promoting healthy intestinal
flora (Gropper and Others 2009).
5
Blackberries are also known to be high in anthocyanin content and antioxidant
capacity (Puupponen-Pimia and Others 2005; Tsuda 2012). Consumption of
anthocyanins has been demonstrated to assist with obesity and diabetes prevention and
or control (Tsuda 2012). Cyanidin-3-glucoside (C3G) is an anthocyanin found in
blackberries. Consumption of this chemical has been shown to decrease body fat
accumulation in mice even when high fat meals were consumed. The mechanism
proposed involved a decrease in lipid synthesis in the liver and white adipose tissues
(Tsuda 2012). Cyanidin-3-glucoside was also found to upregulate the expression of
adiponectin, which is associated with increased insulin sensitivity (Tschriter 2003; Tsuda
2012).
Metabolic syndrome is also associated with increased inflammation, and
anthocyanins were associated with the reduction of the inflammatory markers, tumor
necrosis factor-α and monocyte chemotactic protein-1 (Sasaki and Others 2007; Tsuda
2012). Gallic acid is a metabolite of anthocyanins and is a powerful antioxidant (Yao and
Others 2004). Antioxidants can scavenge free radicals and prevent damage to proteins,
lipids, and DNA within the human body. Gallic acid is present in blackberries, but is also
a byproduct of anthocyanin metabolism by microorganisms in the human
gastrointestinal tract (Aura and Others 2005; Kempler and Humpf 2005).
Blackberries
can be a healthy addition to the diet considering the numerous ways that they can
positively impact human health.
6
1.3 Potential microbial Risks
To our knowledge, blackberries have never been implicated in a food safety
incident; however microbial safety risks may exist. While chemical and physical hazards
are always a possibility (e.g., improperly applied pesticides, thorns), by and large, the
most important potential hazards are biological in nature. Sources of microbial
contamination could include, but are not limited to: birds, insects, and other animals in
the field, microorganisms in soil, environmental molds, contaminated irrigation water,
and humans practicing poor hygiene.
Machine harvested fruit has less human contact than hand-harvested fruit.
However, the impact of proper sanitation of mechanical harvesters is unknown.
Additionally, it is possible for the harvester to disturb soil resulting in aerosols, an issue
if the soil were contaminated with pathogenic microorganisms. This would be of
particular concern when using improperly composted manure in the production system,
as it has been found to be able to harbor E. coli for extended periods of time (Beuchat
2002).
Fruit that is destined for the fresh market is hand harvested. The safety of this
fruit depends on the hygiene practices of the workers. Hand-harvested fruit is packed
directly into the plastic clamshell containers that are sold in fresh markets. The fruit
does not undergo any washing or microbial decontamination step before reaching the
consumer. The clamshells are handled in a way to prevent them from becoming
7
contaminated, using boxes on elevated carts to store them so that they do not touch
the ground (Strik and Others 2007). Additionally, fruit that is visibly moldy or in contact
with the ground is not harvested (personal observations).
Safety becomes an even larger concern when considering that during the offseason, blackberries for fresh market and frozen use are often imported to the U.S. from
Mexico and Guatemala (Strik and Others 2007). Guatemalan raspberries (genus, Rubus)
were found to be the source of several Cyclosporiasis outbreaks, eventually leading to
them having a Detention Without Physical Examination (DWPE) Import Alert placed on
them (Herwaldt and Others 1997; FDA 2012). This has resulted in Guatemalan
raspberries being prohibited from import during the months of March-August every
year (FDA 2012). The DWPE only applies to raspberries; blackberries from Guatemala
are allowed to be imported. Frozen raspberries have also been implicated in hepatitis A
and norovirus outbreaks (Falkenhorst and Others 2005; Reid and Robinson 1987). At
the time of this writing, frozen berries are being implicated in a hepatitis A outbreak
that is affecting Denmark, Norway, Sweden, and Finland. At this time, the type of berry
and origin are unknown (Gillesberg Lassen 2013). The same strain of hepatitis A has
surfaced in an outbreak currently affecting the United States. Again, a frozen berry
blend is believed to be the source with the investigation focusing on imported
pomegranate seeds that were part of that blend (CDC 2013; Terry 2013). Although not
in the Rubus genus, Strawberries (genus Fragaria) are another berry that has been
associated with foodborne illness. They were found to be the vehicle for a 2011
8
outbreak of E. coli O157:H7 in Oregon that left one person dead and several more ill: an
outbreak later found to be due to deer feces being present in the strawberry fields (FDA
2011; Stone 2011; Terry 2011).
Raspberries and blackberries have a similar anatomy. They are comprised of
small structures called drupelets that are held together by small hairs that intertwine
(Bowling 2000). Both have what is called a receptacle, where the fruit is attached to the
plant. Blackberries detach above this and are harvested with the receptacle still in
place, which is seen as the white center when looking at the top of a blackberry.
Raspberries leave the receptacle behind when harvested, which leaves a hollow center
in the fruit (Bowling 2000).
The anatomy of the blackberry could potentially give microorganisms of health
concern the ability to contaminate and survive on fruit, even if later washing was
implemented. The berry skin may not be an ideal location for microorganisms to adhere
due its hydrophobic nature; however, the many crevices that exist where drupelets
meet, and the receptacle, may provide niches for microorganisms to survive (Bowling
2000). This would be a concern largely for fresh market blackberries, but also exists for
the minimally processed IQF blackberries, since many microorganisms can survive
freezing. Other blackberry products may not be as susceptible due to the processing
method. Puree is often pasteurized, and then frozen, and juice is required to be
pasteurized before reaching the consumer. Jams and jellies are heated to an extent that
would kill any vegetative bacterial cells. The hostile environment of fermentation
9
during the berry wine making process would be unfavorable to bacterial pathogens,
particularly when coupled with the dominance of yeast in the system. The concern with
these further processed products is the potential for pathogenic bacteria to be able to
survive if post processing contamination occurred. There is evidence that frozen purees
and juice concentrates from various fruits can support the survival of E. coli O157:H7,
Salmonella spp., and Listeria monocytogenes for at least 12 weeks (Oyarzabal and
others 2003). Additionally, E. coli O157:H7, Salmonella spp, and Listeria innocua were
able to survive on the surface of frozen strawberries and in strawberry juice over various
periods of storage time (Duan and Zhao 2009; Knudsen and Others 2001).
Aside from bacterial contamination, blackberries, like other agricultural
products, have an opportunistic microflora that will spoil harvested fruit thus, limiting
shelf-life. The naturally occurring microflora largely consists of yeasts and molds and
will cause spoilage over time. Many fungal species can survive well in the low pH
environment of blackberries and consume the organic acids present, which can lead to
an increase in the pH of their environment (Tournas and Katsoudas 2005). Depending
on the rate at which that occurred, it could be possible for the pH to rise enough to
allow some of the more acid tolerant bacterial pathogens to be able to perhaps survive
(e.g., E. coli and Salmonella spp.). In retail samples taken from the Washington, D.C.
metro area, 100% of blackberries had some sort of fungal population with 78%
containing Botrytis cinerea, 33% containing Cladosporium, 22% Fusarium, 22% Penicillin,
and 11% Rhizopus (Tournas and Katsoudas 2005).
10
1.4 Potential Antimicrobial Properties
Blackberries and blackberry products have many components that may have
various degrees of antimicrobial activity. Blackberries naturally have a low pH that
ranges from 3.0 to 4.2 (Beuchat 1987). The organic acids present in blackberries are
predominantly citric and malic acids. These organic acids can exist in a variety of ratios,
depending on cultivar (Fan-Chiang 1999). Organic acids exist in their undissociated form
at low pH, which allows them to permeate the plasma membrane of microorganisms
(Brul and Coote 1999). Once inside the cell, the organic acids can then dissociate and
cause a buildup of protons, eventually becoming toxic to the microorganism (Brul and
Coote 1999).
Essential oils have been found to be effective against some microorganisms. For
example, geraniol, found in blackberries (Du and Others 2010), has been shown to have
antimicrobial activity against Salmonella enterica, E. coli O157:H7, and L. innocua
(Friedman and Others 2004; Raybaudi-Massilia and Others 2006).
Blackberries are a rich source of phenolic compounds including phenolic acids,
anthocyanins, and ellagitannins (Puupponen-Pimia and Others 2001; Ruyi and Others
2010). Phenolic compounds may also contribute to antimicrobial activity. Phenolic
acids have been demonstrated to be effective against Gram-negative bacteria
(Puupponen-Pimia and Others 2001). Some of the phenolic acids and flavonoid
compounds found in blackberries include: gallic acid, caffeic acid, coumaric acid, ferulic
11
acid, ellagic acid, catechin, quercetin, and myricetin (Sellappan and Others 2002; Bilyk
and Sapers 1986). While tannins may not directly act against microorganisms, they may
act indirectly by binding substances needed for their survival such as nutrients, and
interfering with microbial extracellular enzyme functions (Puupponen-Pimia and Others
2005). Nohynek and Others (2006) found that extracts from berries in the genus Rubus,
cloudberry and raspberry, were able to permeabilize and in some cases disintegrate the
outer membrane of Gram-negative bacteria. They attributed this to a synergistic effect
of pH and phenolic compounds, noting that gallic acid appeared to have the greatest
effect which they believed was due to its ability to chelate divalent cations from the
membrane, destabilizing it (Nohynek and Others 2006). This trait is also shared by citric
acid (Brul and Coote 1999). In addition, the extracts were found to have an inhibitory
effect against several Gram-positive microorganisms, including Staphylococcus aureus.
Various wines, and even their unfermented juices, have been demonstrated to
have bactericidal properties. Trials with chardonnay juice resulted in bacterial
populations of E. coli O157:H7 and Salmonella Typhimurium being reduced to
undetectable levels between 3-12 days, and the corresponding wine between 10-57
minutes; pinot noir juice between 10-16 days, and 10-60 minutes in the wine (Just and
Daeschel 2003). Another study with unknown grape varieties found that white and red
wines resulted in the reduction of E. coli O157:H7 and Salmonella Enteritidis to
undetectable levels within 30 minutes (Sugita-Konishi and Others 2001). Interestingly,
they did not associate the ethanol content of the wine with the lethality observed due
12
to a solution of 14% ethanol in phosphate-buffered saline not resulting in any reduction
in the bacterial populations over a 60 minute period. That conclusion may have
neglected to account for the synergistic effect of ethanol, organic acids, and phenolic
compounds acting within the same system. Ethanol is known to have a solubilizing
effect on the membranes of bacteria, which may allow for easier permealization of
other constituents that are toxic to the bacterial cell (Willey and Others 2008).
Blackberries are harvested with a soluble solid content far below what is
standard for wine grapes (~10-11 °Brix versus 24°Brix) (Du and Others 2010; Zoecklein
and Others 1990). Blackberry wines would naturally result in a lower ethanol content
than grape wines due to the reduced sugar content unless supplemented. The effect on
lethality that this would have in a blackberry wine fermented without sugar
supplementation has not, to our knowledge at this time, been previously investigated.
While the ethanol content of blackberry wines would be lower, the pH would not be
dissimilar, yet the phenolic content would vary widely, depending on fruit type and
cultivar.
13
2. An Evaluation of Blackberry Harvest Sanitation and the Ability of Escherichia coli
O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus
aureus to Survive on the Surface of Fresh and Frozen Blackberry Fruit
Melissa M. Sales and Mark A. Daeschel
Oregon State University
Department of Food Science and Technology, Corvallis OR 97331
To be submitted to:
Journal of Food Science
Institute of Food Technologists
525 W. Van Buren Ste 1000
Chicago Ill 60607
14
2.1 Abstract
Blackberries, genus Rubus, are an important Oregon agricultural commodity. In
their fresh and processed forms, they offer many health benefits and may be able to
help Americans better achieve fruit consumption recommendations because of
convenience and pleasant sensory qualities. However, the susceptibility of blackberry
products to contamination with bacterial pathogens of human health concern is
unknown. Blackberries have never directly been implicated in a food safety incident;
however, raspberries, also in the Rubus genus, have been the vehicle for hepatitis A,
norovirus, and Cyclospora cayetanensis outbreaks. Furthermore, strawberries were
recently the source of an Escherichia coli O157:H7 outbreak in Oregon.
To better understand the potential for microbial pathogen contamination and
the ability of these organisms to survive or grow in blackberry products, several studies
were conducted. Fresh berries from the ‘Obsidian’ and ‘Triple Crown’ cultivars were
evaluated at different harvest periods for the aerobic plate count, coliforms, yeasts, and
molds to establish a baseline microbial population. Environmental samples were taken
from a clean mechanical harvester and then from the same harvester that had been
intentionally left soiled with berry harvest debris to determine the impact of harvester
sanitation. Samples from ‘Marion’ and ‘Black Diamond’ cultivars were hand harvested
and evaluated for E. coli O157:H7 and Salmonella spp. by rapid detection methods via
the NEOGEN® Reveal® 2.0 systems. Fresh, wild ‘Himalaya’ blackberries and frozen
blackberries from the ‘Triple Crown’ cultivar were spot inoculated with E. coli O157:H7,
15
Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus to
determine the ability of these microorganisms to survive on the berry surface.
‘Himalaya’ samples were stored for 3 days at ambient temperatures and ‘Triple Crown’
for 6 months at -23.3°C.
Aerobic plate counts (APC) for ‘Obsidian’ and ‘Triple Crown’ cultivars ranged
from 3.52-4.62 log CFU/g of berry with the late harvest ‘Triple Crown’ samples having a
significantly higher APC than early harvest samples (p = 0.005). Yeasts and molds
ranged from 3.01-4.73 log CFU/g berry with both cultivars having significantly higher
counts at later harvest times (p = 0.048 ‘Obsidian’; p < 0.001 ‘Triple Crown’). Coliforms
were detected in ‘Obsidian’ mid-harvest and ‘Triple Crown’ early-harvest samples at
2.10 and 1.40 log CFU/g of berry, respectively. The overall aerobic plate counts
measured from the mechanical harvester were not affected by machine cleanliness.
Escherichia coli O157:H7 and Salmonella spp. were not detected using rapid detection
methods in evaluated ‘Marion’ and ‘Black Diamond’ samples.
Escherichia coli O157:H7 was not detectable in fresh or frozen inoculated
samples. Salmonella Typhimurium was detected in 2 frozen samples with 2.95 and 3.21
log reductions. Listeria monocytogenes was only detected in frozen samples and
experienced log reductions ≥ 2.42. Staphylococcus aureus was detectable on every
fresh and frozen berry inoculated with log reductions ranging from 0.67 to 3.48, the
greatest reductions occurring in fresh samples.
16
The overall results suggest that blackberries are not an ideal environment for E.
coli O157:H7, Salmonella Typhimurium, L. monocytogenes, and S. aureus to grow.
However, these microorganisms may be able to survive depending on the type of
blackberry product and its subsequent storage.
2.2 Introduction
Blackberries, genus Rubus, are an important agricultural commodity in Oregon.
In 2011, 24,212,760 kg of cultivated blackberries were produced in Oregon and valued
at over 43 million USD (USDA 2012). There are three types of blackberries plants:
trailing, erect, and semierect. Oregon is unique in that the majority of cultivated
blackberries in this state are trailing cultivars, most notably ‘Marion’, whereas many
other growing regions cultivate erect and semierect types (Strik and Others 2007; USDA
2012). Trailing cultivars are typically machine harvested for processed markets, while
erect and semierect cultivars are hand-harvested for fresh market. There are
exceptions, and the popular ‘Black Diamond’ cultivar is an example. ‘Black Diamond’ is a
trailing cultivar, but has the benefit of being thornless and produces fruit firm enough to
be able to withstand shipping and handling required for fresh market. It can be
mechanically or hand-harvested depending on its intended use (Finn and Others 2005;
Strik and Others 2007). Machine harvested fruit is most often destined for further
processing into individually quick frozen (IQF) berries, jams, purees, juices, and even
wines (Strik and Others 2007). The trailing cultivar ‘Obsidian’ is hand-harvested for
fresh market (Finn and Others 2005). ‘Triple Crown’ is a popular semierect cultivar in
17
Oregon and is hand harvested for fresh market (Finn and Strik 2008; Strik and Others
2007). ‘Himalaya’ is considered an invasive species, but is still popular for noncommercial harvesting in Oregon (Finn and Strik 2008).
Table 2.1 Cultivar Information
Cultivar
‘Marion’
‘Black Diamond’
‘Obsidian’
‘Triple Crown’
‘Himalaya’
Type
Trailing
Trailing
Trailing
Semierect
N/A (similar to semierect)
Farming Method
Certified Organic
Certified Organic
Certified Organic
Certified Organic
Wild
Blackberries are harvested from June through October, depending on the
cultivar and type, with each cultivar having a fruiting season of 3-6 weeks (Finn and Strik
2008; Strik and Others 2007). When considering the value of the Oregon blackberry
harvest combined with the short fruiting season, the economic impact of a food safety
recall could devastate the industry. The season is of such short duration that economic
recovery following a recall would be very unlikely.
Blackberries have never been implicated in a food safety incident; however,
other berry fruits have. Frozen raspberries, also in the Rubus genus, have been the
source of Cyclospora cayetanensis, norovirus, and hepatitis A outbreaks (Ho and Others
2002; Reid and Robinson 1987; Sarvikivi and Others 2012). The anatomy of raspberries
is similar enough to blackberries to warrant concern of blackberries being susceptible to
similar contamination. Currently, frozen berries are believed to be the source of a
hepatitis A outbreak in Denmark, Norway, Sweden, and Finland (Gillesberg and Lassen
18
2013). The same strain of hepatitis A has surfaced in an outbreak affecting the United
States, again a frozen berry blend believed to be the source with the investigation
focusing on imported pomegranate seeds that were included in the blend (CDC 2013;
Terry 2013). Although not in the Rubus genus, fresh strawberries (genus Fragaria) were
the source of an outbreak of Escherichia coli O157:H7 in Oregon that left one dead and
several others ill, demonstrating that other berry fruits are vulnerable to contamination
as well (FDA 2011; Terry 2011).
The outbreaks associated with raspberries and other berry blends have in
common that humans are the reservoir for hepatitis A, Cyclospora cayetanensis, and
norovirus. This suggests that berries may be susceptible to contamination with other
causal agents of foodborne illness that humans are known to be carriers of. These could
include Staphylococcus aureus, pathogenic strains of Escherichia coli, and Salmonella
spp. Thus, poor hygiene of workers involved with harvesting and/or handling fruit could
result in contamination of blackberries.
Other sources of contamination are possible. Birds are known reservoirs of
Salmonella. Flies have been demonstrated to be able to transmit E. coli O157:H7 to
damaged apples (Janisiewicz and Others 1999). Deer were determined to be the source
of the E. coli O157:H7 outbreak associated with strawberries (Stone 2011; Terry 2011).
Additionally, contaminated soil or irrigation water, and perhaps even the harvesting
equipment could be sources of contamination.
19
Blackberries are a portable, convenient fruit with a pleasant aroma and flavor
that could encourage individuals in the United States to consume the recommended
daily servings of fruits (Du and Others 2010). Nationally, only 32.5% of Americans
consume at least two servings of fruit daily (CDC 2010). Increasing fruit and vegetable
consumption as a strategy to combat the obesity epidemic is a goal of the Centers for
Disease Control (CDC 2011). These goals increase the need to understand how
foodborne pathogens behave in various fruit and vegetable environments to avoid
increased consumption having unintended consequences. Understanding how certain
microorganisms behave in the blackberry environment will help the blackberry industry
better employ prevention strategies, understand the risk involved with their product,
and to be prepared to quickly respond in the event of any contamination.
Blackberries are known to naturally have a diverse microflora of yeasts and
molds on their surface (Tournas and Katsoudas 2005); however, not much information
that quantifies the microflora is available. The relationship that these yeasts and molds
may have with bacterial pathogens, if present, is not well understood. It has been
suggested that they may inhibit pathogenic species by dominating the environment or
may even encourage growth by making nutrients available and altering the surrounding
pH (Beuchat 2002; Tournas and Katsoudas 2005).
Blackberries have chemical constituents that could affect the ability of
foodborne pathogens to survive. Organic acids are abundant in blackberries, resulting
in the fruit having a low pH; conditions known to prevent growth and cause bacterial
20
death in many species (Beuchat 1987; Brul and Coote 1999; Jay and Others 2005).
Blackberries are also a rich source of phenolic compounds, many of which have been
found to be bactericidal. Phenolic acids have been shown to be effective against Gramnegative bacteria and tannins are known to bind substances the bacterial cells need to
survive (Puupponen-Pimia and Others 2005, 2007). Furthermore, extracts from other
berries in the Rubus genus have been found to be antagonistic toward Gram-negative
and Gram-positive bacteria (Nohynek and Others 2006).
To better understand the microflora associated with blackberries and how
pathogenic bacteria may behave in whole berries, three primary goals for this study
were established: 1) establish quantifiable information about the microflora populations
on blackberries; 2) evaluate the sanitation of mechanical harvesters; and 3) evaluate the
ability of foodborne pathogens to survive on fresh and frozen blackberries.
2.3 Materials and Methods
2.3.1 Fresh Field Samples
Samples of ‘Obsidian’ and ‘Triple Crown’ blackberries at their early, mid,
mid/late, or late harvest periods were obtained from Riverbend Organic Farms in
Jefferson, OR and evaluated for the aerobic plate count (APC), yeast and molds, and
coliforms. Samples arrived in plastic clamshell containers and were analyzed the same
day as harvest. Two clamshells were obtained for each cultivar at each harvest point
evaluated. A 50 g sample taken from each clamshell was homogenized at 200 RPM for 2
minutes (Stomacher® 400 Circulator; Seward Laboratory). Homogenized samples were
21
immediately serially diluted in sterile Butterfield’s phosphate buffer (referred to as
phosphate buffer for simplicity) and plated on Plate Count Agar (PCA;Difco), Dichloran
Rose Bengal Chloramphenicol Agar (DRBC; EMD), and Violet Red Bile Agar (VRB; ColeParmer Instrument Company). Plate Count Agar plates were incubated for 48h at 30°C,
DRBC plates for 48-72h depending on growth at 30°C, and VRB plates for 24h at 35°C.
2.3.2 Direct Pathogen Testing
Early, mid, and late harvest samples of ‘Marion’ and ‘Black Diamond’
blackberries were collected by hand-harvesting at the North Willamette Research and
Extension Center (NWREC) in Aurora, OR. Berries were separated by cultivar and placed
in plastic bags. They were transported under refrigeration to Oregon State University
and evaluated the same day. Samples were evaluated for Salmonella spp. and E. coli
O157:H7 using modified protocols for the NEOGEN® Reveal® 2.0 Salmonella and
NEOGEN® Reveal® 2.0 E. coli O157:H7 Complete Systems for rapid detection.
From each cultivar, 25 g samples were placed in a sterile 500 ml media bottle
and covered with 100 ml of phosphate buffer. This was conducted in duplicate for each
cultivar. The berries were gently agitated in the solution at room temperature.
For Salmonella testing, one bottle of REVIVE® recovery medium was added to a
sterile 500 ml media bottle and mixed with 200 ml of sterile deionized (DI) water at 4243°C. From the berry sample, 25 ml of phosphate buffer was removed, added to the
REVIVE® mixture and then incubated for 4 h at 36°C. The contents of one bottle of
provided 2xRV selective media was added to 200 ml of sterile DI water at 37°C and then
22
held at 42°C until ready to use. After the solution containing the sample completed its 4
h incubation, the 2xRV solution was added to it and then incubated at 42°C for an
additional 24 h. Using the provided sterile dropper, 200 µl were transferred to a sample
cup. The test strip was inserted and allowed to stand for 15 m at room temperature
before reading.
For E. coli testing, one bottle of Reveal® 2.0 E. coli O157:H7 media was placed in
a sterile 500 ml media bottle and mixed with 325 ml of sterile DI water at 42°C. From
the berry sample, 65 ml of the phosphate buffer was removed, added to the media, and
incubated at 42°C for 20 h. After incubation, 200 µl was transferred to the test cup
provided. A drop of provided promoter agent was added and then incubated for an
additional 15 m. The test strip was placed in the sample cup while it was still in the
incubator and was read after 15 m.
All media and reagents used were included in the test kits and experiments were
conducted in duplicate. All incubation periods occurred with media bottles loosely
lidded to allow for air exchange. Efficacy of the NEOGEN® Reveal® systems was verified
using pure culture spiked berry samples and negative controls.
2.3.3 Mechanical Harvester
Sterile swabs were used to take 8 environmental samples from a clean
mechanical harvester (over-the-row Littau Harvester; Stayton,OR) at the NWREC just
prior to harvesting ‘Marion’ blackberries. Locations sampled were photographed for
future reference and can be viewed in Appendix II. Swabs were moistened in phosphate
23
buffer prior to location sampling. Once sampled, the swabs were agitated in culture
tubes containing 4 ml of phosphate buffer. Samples from the tubes were serially diluted
and plated on PCA. Plates were incubated for 48 h at 30°C.
The mechanical harvester remained intentionally unwashed after the ‘Marion’
harvest and post-harvest environmental samples were taken 48 h after the initial
samples. The same 8 locations were sampled. Sample handling and plating occurred in
the same manner as the initial sampling.
2.3.4 Whole Berry Inoculation
2.3.4.1 Culture Preparation
Cultures of Escherichia coli O157:H7 (ATCC 43894), Salmonella Typhimurium
(ATCC 14028), Listeria monocytogenes (Scott A), and Staphylococcus aureus (general
food isolate, 648 in OSU Culture Collection) were used for spot inoculation studies. All
cultures used were from the culture collection at Oregon State University, Department
of Food Science and Technology. Cultures maintained in Trypticase Soy Broth (TSB)
were transferred to fresh TSB and incubated for 24h at 35°C (BBL™ Trypticase Soy
Broth™, BD). One hundred microliters from each were again transferred to fresh TSB
and incubated for 18 h at the same temperature. These cultures were then serially
diluted in phosphate buffer and used for inoculation. Enumeration was determined by
serial dilution on Trypticase Soy Agar (TSA) (Bacto™ Agar, BD; BBL™ Trypticase Soy
Broth™, BD).
24
2.3.4.2 Fresh Berry Samples
‘Himalaya’ blackberries were obtained from Bald Hill Vineyard in Corvallis, OR and
kept at 8°C. Inoculation occurred the day following harvest. Individual berries were
placed on a sterile cap, which were then placed in a Nalgene tray. Berries were then
inoculated with 10 µl aliquots of a single bacterial strain in 5 locations for a total of 50 µl
per berry (5.18-6.47 log CFU/g of berry). Inoculated berries were allowed to dry
overnight under a biosafety hood. After drying, the tray containing the berries was
moved to an incubator held at room temperature and rotated several times per day for
3 d.
Each berry was then placed in a beaker, had 10 ml of phosphate buffer added to
it and then gently agitated. This ‘rinse’ solution was then serially diluted and plated on
TSA. All plates were incubated at 35°C for a period of 6 h to allow injured cells an
opportunity to recover (Mahmoud and Others 2010). Using the weight of the berry
sample, enough phosphate buffer was added to the berry/phosphate sample to equal a
1:10 dilution and then homogenized at 230 RPM for 30 s (Stomacher®400 Circulator,
Seward). The resulting mixture was serially diluted and plated on TSA, allowing for the
same recovery period as described above. After the recovery period, a layer of
appropriate selective media was placed over the TSA: Sorbitol MacConkey Agar(SMAC;
Difco) for E. coli O157:H7; Xylose Lysine Deoxycholate Agar (XLD; Difco) for Salmonella
Typhimurium; Oxford Listeria Agar Base with Oxford Listeria Selective Supplement (EMB
25
and Sigma Aldrich) for L. monocytogenes; and Mannitol Salt Agar (MSA; BBL) for S.
aureus.
2.3.4.3 Frozen Berry Samples
‘Triple Crown’ blackberries were hand harvested from Riverbend Organic Farms
and kept at 8°C. Inoculation occurred the day following harvest. Individual berries were
placed in sterile glass jars and inoculated with a single bacterial strain (3.54-4.55 log
CFU/g of berry). Berries were inoculated with 5 µl aliquots in 10 locations for a total of
50 µl per berry. Berries were allowed to remain under a biosafety hood until visibly dry,
covered with sterilized foil, and frozen at -23.3°C for 6 months. For evaluation, each
berry was removed from the freezer just prior to analysis. Evaluation occurred in the
same manner as described above for fresh berry evaluation with the following
modification: initial rinse with 10 ml phosphate buffer occurred in the glass jar
containing the berry, after which the berry was gently removed with forceps and placed
into a filtered stomacher bag.
2.3.5 Data Analysis
All data were analyzed using Microsoft Excel with the exception of analyses of
the aerobic plate count, yeasts and molds, and mechanical harvester aerobic plate
counts, which were evaluated by hypothesis tests with Statgraphics Centurion.
2.4 Results and Discussion
2.4.1 Fresh Field Data
The aerobic plate counts (APC) for ‘Obsidian’ and ‘Triple Crown’ cultivars at their
early, mid, mid/late, or late harvest periods ranged from 3.52 to 4.62 log CFU/g of berry
26
(Figure 2.1). Counts were higher for both cultivars at their later harvest time, with
‘Triple Crown’ mid/late harvest having significantly higher APC (p = 0.005). Figure 2.2
shows that yeasts and molds ranged from 3.01 to 4.73 log CFU/g of berry, with
‘Obsidian’ late harvest having the highest values. Later harvests for both cultivars were
observed to have significantly higher yeast and mold counts than earlier harvests (p =
0.048 ‘Obsidian’; p < 0.001 ‘Triple Crown’). Caution should be taken when interpreting
these findings considering that the summer of 2011, when these samples were taken,
experienced an exceptional amount of rain (Oregon Climate Service 2011). This could
have resulted in lower values than normal if microorganisms were being washed off
fruit by the rain, or higher values if the increased moisture was providing better
growth/survival conditions. Published values for the aerobic plate count, yeasts, and
molds for blackberries were not found in a search of the literature; however, our data
were consistent with values obtained from fresh strawberry rinse water (Jensen and
others 2012).
Coliforms were detected in ‘Obsidian’ mid harvest and ‘Triple Crown’ early
harvest samples at 2.10 and 1.40 log CFU/g of berry, respectively (Figure 2.3). The
detection limit for the experimental design was 0.70 log CFU/g. Follow up tests were
not conducted to determine if these coliforms were fecal in origin.
27
APC Log CFU/g
Figure 2.1 Aerobic Plate Count at Various Harvest Times for 'Obsidian' and 'Triple Crown'
Cultivars. Bars indicate the range (high and low values); weeks are for 2011.
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Obsidian Mid
Obsidian Late Triple Crown Early Triple Crown
Harvest: Week 29 Harvest: Week 30 Harvest: Week 34 Mid/Late Harvest:
Week 35
Figure 2.2 Yeasts and Molds at Various Harvest Times for 'Obsidian' and 'Triple Crown'
Cultivars. Bars indicate the range (high and low values); weeks are for 2011.
Yeasts and Molds Log CFU/g
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Obsidian Mid
Obsidian Late Triple Crown Early Triple Crown
Harvest: Week 29 Harvest: Week 30 Harvest: Week 34 Mid/Late Harvest:
Week 35
28
Figure 2.3 Coliforms at Various Harvest Times for 'Obsidian' and 'Triple Crown' Cultivars.
Dotted line indicates the lower limit for the detection of coliforms; Bars indicate the
range (high and low values); weeks are for 2011.
2.5
Coliforms Log CFU/g
2
1.5
1
<0.70 Log CFU/g
<0.70 Log CFU/g
0.5
0
Obsidian Mid
Harvest: Week 29
Obsidian Late
Harvest: Week 30
Triple Crown Early
Harvest: Week 34
Triple Crown
Mid/Late Harvest:
Week 35
2.4.2 Direct Pathogen Testing
The results for the direct testing of E. coli O157:H7 and Salmonella spp. are
shown in Table 2.2. Neither pathogen was detected in any of the samples evaluated.
Table 2.2 Detection of E. coli O157:H7 and Salmonella spp. in ‘Marion’ and ‘Black Diamond’
Cultivars using rapid detection methods
Early 7/9/12
‘Marion’
E. coli
Salmonella
Rep 1
Rep 2
Rep 1
Rep 2
Negative
Negative
Negative
Negative
‘Black
Diamond’
Negative
Negative
Negative
Negative
Mid 7/12/12
‘Marion’
Negative
Negative
Negative
Negative
‘Black
Diamond’
Negative
Negative
Negative
Negative
Late 7/19/12
‘Marion’
Negative
Negative
Negative
Negative
‘Black
Diamond’
Negative
Negative
Negative
Negative
29
2.4.3 Mechanical Harvester
Microbial populations for individual sample locations on the mechanical
harvester varied substantially (Figure 2.4). Little change occurred pre- and post-harvest
at locations 2, 5, 6, and 7. Photographs of sampled locations can be viewed in Appendix
II. A 2.03 log reduction was observed at location 3 post-harvest. Moderate increases
were observed at locations 1, 4, and 8 with location 8 experiencing the largest increase
(2.0 log).
Figure 2.4 Mechanical Harvester Aerobic Plate Counts: Clean and Unwashed
5
4.5
4
Log CFU/Swab
3.5
3
2.5
Clean
2
48 h After Intentionally Left
Unwahsed
1.5
1
0.5
0
Location on Mechanical Harvester
When an overall aerobic plate count for the mechanical harvester was
determined using all locations sampled, there was not a significant difference in clean
30
and intentionally dirtied values (p = 0.452). These results indicate that the microbial
quality of the mechanical harvester does not change 48h post-harvest, even with
washing not occurring between sampling periods. This is likely due to the organic acids
released from the berries during harvest combined with UV radiation inhibiting growth
(Brul and Coote 1999; Rico and Others 2007).
2.4.4 Spot Inoculation Studies
2.4.4.1 Fresh Berry Results
Escherichia coli O157:H7, Salmonella Typhimurium, and L. monocytogenes were
not detected in the ‘Himalaya’ samples 3 d after inoculation (Tables 2.3-2.5).
Staphylococcus aureus was recovered from both inoculated berries, but only in the
evaluation of the homogenized samples (Table 2.6). Staphylococcus aureus
experienced log reductions of 3.03 and 3.48 in these samples.
Table 2.3 ‘Himalaya’ inoculated with E. coli O157:H7, evaluated after 3 d held at ambient
temperatures
Sample
Inoculum per gram of
berry
Rinse Recovery
Homogenized
Recovery
Berry 1
5.74 Log CFU/g
Berry 2
5.69 Log CFU/g
<0.70 Log CFU/g
< 0.70 Log CFU/g
<1.70 Log CFU/g
<1.70 Log CFU/g
31
Table 2.4 ‘Himalaya’ inoculated with Salmonella Typhimurium, evaluated after 3 d held at
ambient temperatures
Sample
Inoculum per gram of
berry
Rinse Recovery
Homogenized
Recovery
Berry 1
5.80 Log CFU/g
Berry 2
5.87 Log CFU/g
< 0.70 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
<1.70 Log CFU/g
Table 2.5 ‘Himalaya’ inoculated with L. monocytogenes, evaluated after 3 d held at ambient
temperatures
Sample
Inoculum per gram of
berry
Rinse Recovery
Homogenized
Recovery
Berry 1
6.25 Log CFU/g
Berry 2
6.47 Log CFU/g
< 0.70 Log CFU/g
< 0.70 Log CFU/g
<1.70 Log CFU/g
<1.70 Log CFU/g
Table 2.6 ‘Himalaya’ inoculated with S. aureus, evaluated after 3 d held at ambient
temperatures
Sample
Inoculum per gram of
berry
Rinse Recovery
Homogenized
Recovery
Berry 1
5.33 Log CFU/g
Berry 2
5.18 Log CFU/g
< 0.70 Log CFU/g
< 0.70 Log CFU/g
2.30 Log CFU/g
1.70 Log CFU/g
2.4.4.2 Frozen Berry Results
Escherichia coli O157:H7 was not detected in any of the frozen ‘Triple Crown’
samples (Table 2.7). Salmonella Typhimurium was detected in 2 of the 7 inoculated
berries, and only in the rinse water with 2.95 and 3.21 log reductions (Table 2.8). It
32
should be noted that one of the berries was evaluated at 3 months to establish to the
procedure protocol. Listeria monocytogenes was detected in 3 of the 7 inoculated
berries, all 3 in the homogenized samples with recovery also occurring in one of the
rinse samples (Table 2.9). Log reductions of L. monocytogenes ranged from 2.42-3.42
when detected. Staphylococcus aureus was detected in all 7 inoculated berries, 4 of the
7 in the rinse water and 5 of the 7 in homogenized samples (Table 2.10). Log reductions
of S. aureus ranged from 0.67-3.48. The detection limit for rinse water was 0.70 log
CFU/g berry and 1.70 log CFU/g berry for homogenized samples.
Table 2.7 Frozen ‘Triple Crown’ inoculated with E. coli O157:H7, evaluated after 6 months stored
at -23.3°C
Sample
Berry 1
Inoculum per
gram of berry
4.40 Log CFU/g
Rinse Recovery
<0.70 Log CFU/g
Homogenized
Recovery
<1.70 Log CFU/g
Berry 2
4.50 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 3
4.45 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 4
4.42 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 5
4.34 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 6
4.36 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 7
4.31 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
33
Table 2.8 Frozen ‘Triple Crown’ inoculated with Salmonella Typhimurium, evaluated after 6
months stored at -23.3°C (*Berry 1 evaluated after 3 months)
Sample
Rinse Recovery
Berry 1*
Inoculum per
gram of berry
3.95 Log CFU/g
1.00 Log CFU/g
Homogenized
Recovery
<1.70 Log CFU/g
Berry 2
3.90 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 3
4.04 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 4
3.96 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 5
3.91 Log CFU/g
0.70 Log CFU/g
<1.70 Log CFU/g
Berry 6
3.87 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 7
4.00 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Table 2.9 Frozen ‘Triple Crown’ inoculated with L. monocytogenes, evaluated after 6 months
stored at -23.3°C
Sample
Berry 1
Inoculum per
gram of berry
4.32 Log CFU/g
Rinse Recovery
<0.70 Log CFU/g
Homogenized
Recovery
1.70 Log CFU/g
Berry 2
4.42 Log CFU/g
1.00 Log CFU/g
2.00 Log CFU/g
Berry 3
4.24 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 4
4.31 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 5
4.55 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
Berry 6
4.24 Log CFU/g
<0.70 Log CFU/g
1.70 Log CFU/g
Berry 7
4.27 Log CFU/g
<0.70 Log CFU/g
<1.70 Log CFU/g
34
Table 2.10 Frozen ‘Triple Crown’ inoculated with S. aureus, evaluated after 6 months stored at 23.3°C
Sample
Berry 1
Inoculum per
gram of berry
3.59 Log CFU/g
Rinse Recovery
<0.70 Log CFU/g
Homogenized
Recovery
2.18 Log CFU/g
Berry 2
3.62 Log CFU/g
0.70 Log CFU/g
<1.70 Log CFU/g
Berry 3
3.58 Log CFU/g
0.70 Log CFU/g
<1.70 Log CFU/g
Berry 4
3.56 Log CFU/g
1.00 Log CFU/g
1.70 Log CFU/g
Berry 5
3.60 Log CFU/g
<0.70 Log CFU/g
2.93 Log CFU/g
Berry 6
3.54 Log CFU/g
<0.70 Log CFU/g
2.00 Log CFU/g
Berry 7
3.63 Log CFU/g
<0.70 Log CFU/g
1.70 Log CFU/g
These results indicate that the surface of blackberry fruit is not suitable for these
bacteria to grow; however, they may survive. Escherichia coli O157:H7 appears to be
the least able to survive either on fresh or frozen berry surfaces, whereas S. aureus was
the most able to persist. Freezing of the inoculated fruit appears to improve survival,
which was to be expected considering that freezing is a method frequently used to
preserve microorganisms (Jay and Others 2005). Furthermore, E. coli O157:H7 and
Salmonella spp. have been found to be able to survive on the surface of frozen
strawberries for at least 30 d (Knudsen and Others 2001).
The aqueous nature of the inocula may have also contributed to the minimal
survival observed. Blackberry fruit has a waxy surface that protects it from a variety of
stresses, including osmotic pressure (Bowling 2000; Shepherd and Griffiths 2006). It was
35
observed during the inoculation procedure that the inoculum would form beads on the
hydrophobic berry surface. This may have resulted in some of the bacterial cells
experiencing desiccation as the phosphate buffer evaporated, leaving few viable cells in
contact with the actual berry. In harvesting/processing settings, fruit would become
contaminated by feces, hands, soil, etc. These other sources of contamination may lead
to better bacterial survival on the blackberry surface.
2.5 Conclusions
The blackberry surface is not an environment that will allow the growth of E. coli
O157:H7, Salmonella Typhimurium, L. monocytogenes, and S. aureus. The microflora
that was observed in fresh blackberry samples may offer some protection by
outcompeting/antagonizing bacterial pathogens (Beuchat 2002). Moreover, E. coli
O157:H7 was not observed to survive in any fresh and frozen samples within the
experimental detection limits. However, the observation that there was survival by
some pathogens reinforces the need for sanitary harvesting conditions and handling to
prevent the contamination of blackberries that are destined for fresh market or IQF
processing.
The mechanical harvester used to harvest trailing type blackberries is not likely
to be a source of contamination, particularly if it is exposed to sunlight (Rico 2007). Any
blackberry residue may actually reduce microbial populations on the harvester by
exposure to organic acids; however, these results should not be taken as a suggestion to
not wash mechanical harvesters post-harvest. The larger concern lies with the hygiene
36
of workers handling the blackberry fruit. Humans are known reservoirs of S. aureus and
can also harbor pathogenic strains of E. coli and Salmonella spp. Furthermore, they are
also known reservoirs of Hepatitis A, norovirus, and Cyclospora cayetanensis, all of
which have been the cause of foodborne illnesses associated with raspberries indicating
that humans can contaminate berries in the Rubus genus (Ho and Others 2002; Reid and
Robinson 1987; Sarvikivi and Others 2012).
Although E. coli O157:H7 and Salmonella spp. were not detected in directly
harvested and tested samples, it is important to consider other non-human sources of
contamination. Animals, contaminated soil, or irrigation water could result in
contamination with a variety of microorganisms of human concern. These sources can
be avoided by establishing and maintaining sanitary growing, harvesting, and handling
practices.
37
3. An Evaluation of the Survival of Escherichia coli O157:H7, Salmonella Typhimurium,
Listeria monocytogenes, and Staphylococcus aureus in ‘Marion’ and ‘Black Diamond’
Blackberry Juice and Wine
Melissa M. Sales and Mark A. Daeschel
Oregon State University
Department of Food Science and Technology, Corvallis OR 97331
To be submitted to:
Journal of Food Science
Institute of Food Technologists
525 W. Van Buren Ste 1000
Chicago Ill 60607
38
3.1 Abstract
Blackberries, genus Rubus, are an important Oregon agricultural commodity that
is frequently processed into various products. These products include individually quick
frozen (IQF) berries, jams, juices, purees, and even wines. However, the susceptibility of
blackberry products to contamination with bacterial pathogens of human health
concern is unknown. Previous studies and food safety incidents have demonstrated
that many pathogenic microorganisms are able to survive in purees, juices, and frozen
concentrates made from various fruits.
Survival studies were conducted in juices and wines made from ‘Marion’ and
‘Black Diamond’ purees to understand the potential for pathogenic bacteria to survive if
post-processing contamination were to occur. Furthermore, the studies were designed
to yield information about what chemical constituents of the juices and wines may
contribute to antibacterial activity. Escherichia coli O157:H7, Salmonella Typhimurium,
Listeria monocytogenes, and Staphylococcus aureus were evaluated for their ability to
survive in these products.
Growth of microorganisms was not observed in any juice or wine samples.
Maximum observed survival times in juices ranged from 12 h for L. monocytogenes to
108 h for Salmonella Typhimurium. Maximum survival times in wines were 40 m for
both E. coli O157:H7 and Salmonella Typhimurium, and 80 m for both L. monocytogenes
and S. aureus. Adding ethanol to juice samples to equal that of their counterpart wines
decreased survival time for all microorganisms evaluated by several hours. Increasing
39
the pH of wines by approximately one unit increased the survival time from minutes to
hours, and in some cases, days.
These results demonstrate that blackberry juice and wine do not support the
growth of E. coli O157:H7, Salmonella Typhimurium, L. monocytogenes, and S. aureus.
However, these microorganisms may be able to survive for various periods of time
depending on the type of blackberry product. Many constituents of blackberries may
offer bactericidal activity, with organic acids appearing to have the greatest effect.
3.2 Introduction
Blackberries are an important agricultural commodity in Oregon with 65% of the
U.S. production occurring in this state (Strik and Others 2007). There are three types of
blackberry plants: trailing, erect, and semierect. Oregon is unique in that cultivated
blackberries are predominantly trailing types, with the primary cultivar being ‘Marion’,
whereas other growing regions typically grow erect and semierect types (Strik and
Others 2007; USDA 2010). ‘Black Diamond’ is another popular trailing type in Oregon,
but has the added benefit of being thornless and produces firm enough fruit to
withstand shipping and handling, resulting in this cultivar being suitable for both
mechanical and hand-harvesting (Finn and Strik 2008; Strik and Others 2007).
Table 3.1 Cultivar Information
Cultivar
Type
‘Marion’
Trailing
‘Black Diamond’
Trailing
Farming Method
Conventional
Certified Organic
40
Machine harvested blackberries are most often destined for further processing,
whereas hand-harvested fruit is used for fresh market. Machine harvested fruit may be
processed into individually quick frozen (IQF) berries, purees, jams, juices, and even
wines (Strik and Others 2007). Individually quick frozen berries are minimally processed
and could be at risk for contamination. Jams, juices, and some purees undergo a
thermal processing procedure that would effectively kill any vegetative pathogenic
bacterial cells present. These cells would also not be likely to survive fermentation
during the blackberry wine making process. For these fermented and thermally
processed products, the concern is whether or not bacterial pathogens of human
concern could survive in the event of post-processing contamination. The scope of this
study will focus on the ability of foodborne pathogens to survive in processed blackberry
juices and wines.
There is evidence that frozen purees and juice concentrates from various fruits
can support the survival of Escherichia coli O157:H7, Salmonella spp., and Listeria
monocytogenes for at least 12 weeks (Oyarzabal and Others 2003). Previous studies
using chardonnay juice resulted in bacterial populations of E. coli O157:H7 and
Salmonella Typhimurium being reduced to undetectable levels between 3-12 days, and
the corresponding wine between 10-57 minutes; pinot noir juice between 10-16 days,
and 10-60 minutes in the wine (Just and Daeschel 2003). Another study with unknown
grape varieties found that white and red wines resulted in the reduction of E. coli
41
O157:H7 and Salmonella Enteritidis to undetectable levels within 30 minutes (SugitaKonishi and Others 2001).
There are several chemical constituents of blackberry juices and wines that may
contribute to bactericidal activity. Organic acids are abundant in blackberries, typically
as citric and/or malic acids in varying ratios, depending on cultivar (Fan-Chiang 1999).
This results in the fruit having a low pH, conditions known to prevent growth and cause
cellular death in many bacterial species (Beuchat 1987; Brul and Coote 1999; Jay and
Others 2005). Blackberries are also a rich source of phenolic compounds, such as
phenolic acids, anthocyanins, and ellagitannins (Puupponen-Pimia and Others 2001; Wu
and Others 2010). Phenolic acids have been shown to be effective against Gramnegative bacteria and the tannins bind substances the bacterial cells need to survive
(Puupponen-Pimia and Others 2001, 2005). Furthermore, extracts from other berries in
the Rubus genus have been found to be effective against Gram-negative and Grampositive bacteria (Nohynek and Others 2006). Ethanol, present in wines, is known to
have a solubilizing effect on the membranes of bacteria, which may allow for easier
permealization of other constituents that are toxic to the bacterial cell (Willey and
Others 2008).
The objectives for this study were to evaluate the ability of E. coli O157:H7,
Salmonella Typhimurium, L. monocytogenes, and Staphylococcus aureus to survive in
blackberry juices and wines, and to understand which constituents of the juices and
wines contribute to antibacterial activity.
42
3.3 Materials and Methods
3.3.1 Juice and Wine Preparation
Approximately 34 kg each of ‘Marion’ and ‘Black Diamond’ blackberries were
collected for the purpose of making juice and wine. ‘Marion’ fruit was harvested by
machine in a single harvest from the North Willamette Research and Extension Center
(NWREC) in Aurora, OR. After collection, the fruit was pureed and frozen at -23.8°C for
further use. ‘Black Diamond’ blackberries were hand-harvested from Riverbend Organic
Farms in Jefferson, OR. Fruit were harvested over the ‘Black Diamond’ fruiting season
(July, 2011) and frozen at -23.8°C until 34 kg was accumulated, after which, the berries
were thawed and pureed before being frozen again for future use.
Purees were thawed and equally divided. Half of each puree was hand pressed
through a mesh bag in order to release juice. The juices were then bottled in 187 ml
glass bottles, capped with crown caps, and pasteurized in a water bath at 71°C for 25 m.
The other half of each puree was placed into 38 L plastic buckets and inoculated with
Saccharomyces cerevisiae for the purpose of making wine. After 5 d of fermentation at
ambient temperatures, both the liquid and pulp components of the wine were strained
through a mesh bag and the filtered liquids were transferred to 11 L carboys for
continued fermentation for 5 d. The wine was then racked and bottled into 187 ml glass
bottles, crown capped, and pasteurized as described above. Bottles of juice and wine
were boxed and kept out of light at room temperature for further use.
43
3.3.2 Juice and Wine Properties
Sterility of pasteurized juice and wine was determined by ATP bioluminescence
(Firefly®2 Luminometer, Arrow Scientific). The pH and titratable acidity of juice and wine
samples were also measured. Titratable acidity was determined by titration with 0.1N
NaOH to an end point of pH 8.1 and calculated as grams of citric acid/L (pH Meter, ColeParmer). Soluble solid content of purees and juices were determined by refractometry
(RFM 81 Multi Scale Automatic Refractometer, Bellingham Stanley, Inc.). Ethanol
content of wines was determined by ebulliometry (Zoecklein 1990; Dujardin-Salleron
Laboratoires).
3.3.3 Culture Preparation
Cultures of Escherichia coli O157:H7 (ATCC 43894), Salmonella Typhimurium
(ATCC 14028), Listeria monocytogenes (Scott A), and Staphylococcus aureus (general
food isolate, 648 From OSU Culture Collection) were used for juice and wine survival
studies. All cultures used were from the culture collection at Oregon State University,
Department of Food Science and Technology. Cultures were maintained in Trypticase
Soy Broth (TSB) then were transferred to fresh TSB and incubated for 24 h at 35°C (BBL™
Trypticase Soy Broth™, BD). One hundred µl from each were then again transferred to
fresh TSB and incubated for 18 h at the same temperature. These cultures were then
serially diluted in sterile Butterfield’s phosphate buffer (referred to as phosphate buffer
for simplicity) and used for inoculation. Enumeration was determined by serial dilution
on Trypticase Soy Agar (TSA) (Bacto™ Agar, BD; BBL™ Trypticase Soy Broth™, BD).
44
3.3.4 Survival Study Procedure
3.3.4.1 Controls
Due to the presence of pectin, juices were centrifuged at 1613 x g for 5 m prior
to use (IEC Clinical Centrifuge). Of each juice supernatant and wine, 9.9 ml was placed in
culture tubes, to which 100 µl of a single bacterial strain was added. Samples were
serially diluted in phosphate buffer and plated on TSA at appropriate time intervals to
determine the maximum survival time of each bacterium in the control samples.
3.3.4.2 Variables
Juice supernatant samples were adjusted to have an ethanol content that
matched their wine counterpart. For ‘Marion’ juice, 9.405 ml of juice was mixed with
0.495 ml of 100 % ethanol for a total sample volume of 9.9 ml with an ethanol content
of 5%. Of ‘Black Diamond’ juice, 9.484 ml was mixed with 0.416 ml of 100% ethanol for
a total volume of 9.9 ml with an ethanol content of 4.2%. Wine samples were adjusted
with 6M NaOH to increase their pH values by approximately 1 unit in order to evaluate
the impact of pH on bacterial survival. One hundred µl of culture was added to each of
the variable samples and plated in the method described above for control samples.
3.3.5 Data Analysis
All data analyses were conducted using Microsoft Excel.
3.4 Results and Discussion
3.4.1 Properties of Purees, Juices, and Wines
The pH of ‘Marion’ and ‘Black Diamond’ purees, juices, and wines were fairly
similar, ranging from 3.21 to 3.30 for all products (Tables 3.2-4). The soluble solids
45
content of ‘Marion’ puree and juice was slightly higher than ‘Black Diamond’ puree and
juice (Tables 3.2-3). As would be expected based on soluble solids content, ‘Marion’
wine had slightly higher ethanol content than ‘Black Diamond’ wine, at 5.0% and 4.2%,
respectively (Table 3.4). Titratable acidity was higher in the ‘Black Diamond’ juice and
wine than in the ‘Marion’ juice and wine (Tables 3.3-4). These values are consistent
with other reported pH, titratable acidity, and soluble solid content values for
blackberry juice (Vasquez-Araujo and Others 2010).
Table 3.2 pH and Soluble Solid Content of Blackberry Puree
‘Marion’ Juice
‘Black Diamond’ Juice
pH
3.30
3.21
°Brix
12.78
10.88
Table 3.3 pH, Soluble Solid Content, and Titratable Acidity of Blackberry Juices
‘Marion’ Juice
‘Black Diamond’ Juice
pH
3.26
3.24
°Brix
11.73
10.85
TA
13.95 g citric acid/L
14.76 g citric acid/L
Table 3.4 pH, Ethanol Content, and Titratable Acidity of Blackberry Wines
‘Marion’ Wine
‘Black Diamond’ Wine
pH
3.26
3.26
% Ethanol
5%
4.2%
TA
15.85 g citric acid/L
15.99 g citric acid/L
3.4.2 Survival Study Results
Growth was not observed for any of the microorganisms in any of the
treatments. Escherichia coli O157:H7 was observed to no longer be detectable at 84 h
in both ‘Marion’ and ‘Black Diamond’ juices (Figures 3.1 and 3.3). The detection limit for
46
the plating procedure used was 0.70 Log CFU/ml. Salmonella Typhimurium was no
longer detectable at a maximum of 60 h in ‘Marion’ juice and 108 h in ‘Black Diamond’
juice (Figures 3.5 and 3.7). It should be noted that in ‘Black Diamond’ trials, Salmonella
Typhimurium was observed to no longer be detectable at 60 h in one trial, then
remained at or near the detection limit from 60-108 h in the second trial (Figure 3.7).
This may have been the result of a few particularly acid tolerant cells being present.
Staphylococcus aureus survived for 48 and 84 h in ‘Marion’ and ‘Black Diamond’ juices,
respectively (Figures 3.9 and 3.11).
Results for L. monocytogenes tended to be inconsistent among trials, making it
difficult to establish means and ranges. For this reason, results for L. monocytogenes
are reported as maximum survival times in Table 3.5 for all treatments. Listeria
monocytogenes was observed to have the shortest survival time, at 12 h in both juices
(Table 3.5).
Adding ethanol to juice samples had the effect of reducing the survival time of all
microorganisms, often by more than half. The survival time of E. coli O157:H7 was
reduced to 36 h and Salmonella Typhimurium was reduced to 6 h in both juices with
added ethanol (Figures 3.1, 3.3, 3.5, and 3.7). The survival time of S. aureus was
reduced to 24 h and 36 h, L. monocytogenes to 2 h and 1 h in ‘Marion’ and ‘Black
Diamond’ juices with added ethanol, respectively (Figures 3.9 and 3.11, table 3.5). This
reduction in survival time was to be expected due to the ability of ethanol to solubilize
47
the bacterial membrane, making it easier for undissociated organic acids to be able to
penetrate the bacterial cell (Willey and Others 2006).
Escherichia coli O157:H7 was observed to survive for a maximum of 60 m in
‘Marion’ wine and 40 m in ‘Black Diamond’ wine (Figures 3.2 and 3.4). Salmonella
Typhimurium and S. aureus were observed to survive for a maximum of 40 m and 80 m
in both wine samples, respectively (Figures 3.6, 3.8, 3.10, and 3.12). Listeria
monocytogenes survived for a maximum of 80 m in ‘Marion’ wine and 40 m in ‘Black
Diamond’ wine (Table 3.5).
There was a dramatic difference in survival times when comparing the wine to
the juice with added ethanol results, even though both sets of samples had the same
ethanol contents and similar pH values. This may have been due to the juice samples
still having plenty of nutrients, such as carbohydrates and amino acids, available to the
bacterial cells as they attempted to survive the hostile environment of organic acids and
ethanol. The wines would have had many of these nutrients depleted by the yeast
during the fermentation process. Other differences may include the wine containing
additional yeast metabolites that may exhibit antimicrobial activity, such as acetate
(Davison and Stephanopoulos 1986).
Increasing the pH of the wines had the effect of increasing the survival times of
all microorganisms from ≤ 80 m to 6-48 h. In the case of L. monocytogenes, total kill was
not observed; however, a 4.62 log reduction occurred in pH adjusted ‘Marion’ wine and
48
a 2.96 log reduction occurred in pH adjusted ‘Black Diamond’ wine at 48 h (Table 3.5).
In pH adjusted ‘Marion’ wine, E. coli O157:H7 survived for 36 h, Salmonella
Typhimurium for 6 h, and S. aureus for 36 h (Figures 3.1, 3.5, and 3.9). In pH adjusted
‘Black Diamond’ wine, E. coli O157:H7 survived for 48 h, Salmonella Typhimurium for 36
h, and S. aureus for 48 h (Figures 3.3, 3.7, and 3.11).
Increasing the pH of the wines had a dramatic effect on the survival time of all
microorganisms. This was likely due to the increased pH causing more of the organic
acids to exist in their dissociated state, making entry into the bacterial cell more
difficult. The dramatic increase in survival observed with L. monocytogenes is
consistent with another study that found some strains were able to persist over pH 3.5
and 4.0 for several hours (Phan-Thanh and Others 2000).
Figure 3.1 Survival of E. coli O157:H7 in ‘Marion’ Products. Bars indicate the range (high and low
values).
7
Log CFU/ml
6
5
Marion Juice
4
Marion Juice Trial 2
Marion Juice 5% ethanol
3
Marion Wine
2
Marion Wine pH 4.31
1
Detection Limit
0
0
20
40
60
Time in Hours
80
49
Figure 3.2 Survival of E. coli O157:H7 in ‘Marion’ Wine. Bars indicate the range (high and low
values).
7
6
Log CFU/ml
5
4
Marion Wine
3
Marion
WineTrial 2
2
Detection
Limit
1
0
0
20
40
60
Time in Minutes
Figure 3.3 Survival of E. coli O157:H7 in ‘Black Diamond’ Products. Bars indicate the range (high
and low values).
7
6
Black Diamond Juice
Log CFU/ml
5
4
Black Diamond Juice
4.2% ethanol
3
Black Diamond Wine
2
Black Diamond Wine pH
4.14
1
Detection Limit
0
0
20
40
Time in Hours
60
80
50
Figure 3.4 Survival of E. coli O157:H7 in ‘Black Diamond’ Wine. Bars indicate the range (high and
low values).
7
6
Log CFU/ml
5
Black Diamond Wine
4
3
Black Diamond Wine
Trial 1
2
Detection Limit
1
0
0
10
20
30
40
Time in Minutes
Figure 3.5 Survival of Salmonella Typhimurium in ‘Marion’ Products. Bars indicate the range
(high and low values).
7
6
Log CFU/ml
5
Marion Juice
4
Marion Juice Trial 1
3
Marion Juice 5% ethanol
Marion Wine
2
Marion Wine pH 4.31
1
Detection Limit
0
0
20
40
Time in Hours
60
51
Figure 3.6 Survival of Salmonella Typhimurium in ‘Marion’ Wine. Bars indicate the range (high
and low values).
7
6
Log CFU/ml
5
4
Marion Wine
3
Detection Limit
2
1
0
0
10
20
30
40
50
Time in Minutes
Figure 3.7 Survival of Salmonella Typhimurium in ‘Black Diamond’ Products. Bars indicate the
range (high and low values).
Log CFU/ml
7
6
Black Diamond Juice
5
Black Diamond Juice
Trial 1
4
Juice 4.2% Ethanol
3
Black Diamond Wine
2
Wine pH 4.14
1
0
Detection Limit
0
20
40
60
Time in Hours
80
100
52
Figure 3.8 Survival of Salmonella Typhimurium in ‘Black Diamond’ Wine. Bars indicate the range
(high and low values).
7
6
Log CFU/ml
5
4
Black Diamond Wine
3
Detection Limit
2
1
0
0
10
20
30
40
50
Time in Minutes
Table 3.5 Maximum Observed Survival Times of L. monocytogenes in ‘Marion’ and ‘Black
Diamond’ Juices and Wines: All Treatments
Treatment
‘Marion’ Juice
‘Marion’ Juice 5.0% Ethanol
‘Marion’ Wine
‘Marion’ Wine pH 4.31
‘Black Diamond’ Juice
‘Black Diamond’ Juice 4.2% Ethanol
‘Black Diamond’ Wine
‘Black Diamond’ Wine pH 4.14
(Total kill was not observed in pH adjusted wine samples)
Survival Time
12 h
2h
80 m
4.62 Log Reduction in 48 h
12h
1h
40 m
2.96 Log Reduction in 48 h
53
Figure 3.9 Survival of S. aureus in ‘Marion’ Products. Bars indicate the range (high and low
values).
Log CFU/ml
7
6
Marion Juice
5
Marion Juice Trial 1
4
Marion Juice 5.0%
Ethanol
3
Marion Wine
2
Marion Wine pH 4.31
1
0
Detection Limit
0
10
20
30
40
50
60
Time in Hours
Figure 3.10 Survival of S. aureus in ‘Marion’ Wine. Bars indicate the range (high and low values).
7
6
Log CFU/ml
5
4
Marion Wine
3
Marion Wine Trial 1
Detection Limit
2
1
0
-10
10
30
50
Time in Minutes
70
90
54
Figure 3.11 Survival of S. aureus in ‘Black Diamond’ Products. Bars indicate the range (high and
low values).
Log CFU/ml
7
6
Black Diamond Juice
5
Black Diamond Juice
Trial 1
4
Black Diamond Juice
4.2% Ethanol
3
Black Diamond Wine
2
Black Diamond Wine pH
4.13
1
0
Detection Limit
0
20
40
60
80
Time in Hours
Figure 3.12 Survival of S. aureus in ‘Black Diamond’ Wine. Bars indicate the range (high and low
values).
7
6
Log CFU/ml
5
4
Black Diamond Wine
3
Black Diamond Trial 1
Detection Limit
2
1
0
0
20
40
60
Time in Minutes
80
55
3.5 Conclusions
‘Marion’ and ‘Black Diamond’ juices and wines did not support the growth of E.
coli O157:H7, Salmonella Typhimurium, L. monocytogenes, and S. aureus. However,
survival will vary depending on acid and ethanol content. The maximum time that any
of the tested microorganisms was observed to survive was 80 m in the wines and 108 h
in the juices. These findings are consistent with other survival studies conducted using
juice and wine made from wine grapes (Just and Daeschel 2003; Sugita-Konishi and
Others 2001). These results indicate that if blackberry juice or wine became
contaminated post-processing, these particular microorganisms would not be likely to
survive by the time the product reached the consumer. Caution should be taken when
interpreting these findings for juices because these experiments were conducted at
ambient temperatures. If juices were contaminated and held under refrigeration or
freezing conditions, survival may be extended. This could be of major concern for
products such as unpasteurized frozen puree. Further research would need to be
conducted to determine survival times under those conditions.
The constituents of the juices and wines that likely contribute to antibacterial
activity include organic acids, phenolic compounds, and ethanol (wine). These
constituents being present in the same system make it very likely that they act
synergistically. The dramatic increase in survival time that was observed when the pH
of wines was increased by approximately 1 pH unit indicates that the organic acids are a
major antibacterial contributor. Understanding the contribution of each constituent
56
would require conducting survival studies with them individually. Further research
would be necessary to fully understand how the phenolic compounds, in the quantities
that they occur in blackberry juice and wine, affect the ability of the studied bacteria to
survive.
57
4. Overall Conclusions and Future Work
The bacterial pathogens used in these studies were not observed to grow on
fresh and frozen blackberries. Survival was observed for Listeria monocytogenes and
Staphylococcus aureus on fresh, wild ‘Himalaya’ after 3 d and on frozen ‘Triple Crown
after 6 months. Salmonella Typhimurium was detected on frozen ‘Triple Crown’ at 3 and
6 months. These results emphasize the need for sanitary growing, harvesting, and
handling procedures to prevent contamination. Maintaining such procedures will help
ensure that blackberries do not become the center of a food safety incident as
raspberries have in the past (Gillesberg and Lassen 2013; Ho and Others 2002; Reid and
Robinson 1987; Sarvikivi and Others 2012). Many of the causative agents involved with
raspberry recalls are harbored by humans, for example, hepatitis A, norovirus, and
Cyclospora cayetanensis. This indicates that blackberries may be susceptible to
contamination with microorganisms that humans are known reservoirs for including S.
aureus, pathogenic strains of Escherichia coli, and Salmonella spp. in addition to those
previously mentioned. Preventing contamination with these microorganisms requires
emphasis and attention paid to worker hygiene. Furthermore, considering current
recalls, processors are advised to have procedures in place to verify the safety of
imported supplies especially if adding them to berry blends. This could be in the form of
a certificate of acceptance/analysis or letter of guarantee from a foreign supplier of
berry product.
58
Aside from humans, other sources of contamination are possible in the field.
Animals, insects, and contaminated soil or irrigation water could all lead to bacterial
deposits on blackberry fruit (Beuchat 2002; Janisiewicz and Others 1999; Terry 2011).
The mechanical harvester evaluated did not appear to a potential source of
contamination, even when left intentionally unwashed. A diverse microflora was
observed on the surface of fresh blackberries. How this microflora affects the ability of
pathogenic bacteria to adhere to the berry surface and survive is not well understood
and may be worth investigating further.
The inability of E. coli O157:H7, Salmonella Typhimurium, L. monocytogenes, and
S. aureus to survive beyond 80 m in wines and 108 h in juices demonstrate that
blackberries inherently have antibacterial properties. There are several constituents of
blackberry juices and wines that are likely to contribute to bacterial death, including
organic acids, phenolic compounds, and ethanol in the case of wines. These
constituents likely behave synergistically to cause bacterial cells to die. The dramatic
increase in survival time observed when the pH of the wines was increased suggests
that the organic acids are the primary antibacterial constituent. These studies were
conducted at ambient temperatures. Future work should look at what effect the sample
temperature has on survival and should be extended to frozen blackberry puree.
Furthermore, conducting survival studies with isolated phenolic compounds from
blackberries may yield valuable information.
59
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APPENDICES
66
Appendix I. Fresh Field Samples Raw Data, Chapter 2. Bold indicates value used in calculation.
Detection limit calculated as: <1 colony/plate at the lowest dilution with 0.1 ml on each of 2
plates  1/0.2 = 5 CFU/g or 0.70 Log CFU/g.
Table I.1 Aerobic Plate Count Raw Data, Fresh Samples
Rep 1
Dilution on
Plate
10^-3
10^-4
10^-5
10^-6
Plate
1
Plate
2
CFU/g
Log
CFU/g
Plate
1
Plate 2
CFU/g
Log
CFU/g
1
0
0
1
17
0
0
0
9000
3.95
15
5
0
0
31
5
0
0
23000
4.36
Plate 2
CFU/
g
Log
CFU/g
65
70
15
12
0
0
0 Contaminated
6750
3.83
Rep 1
Dilution on
Plate
10^-2
10^-3
10^-4
10^-5
10^-2
10^-3
10^-4
10^-5
Obsidian' Late-Harvest 07/29/11
Rep 2
Plate
1
Plate
2
CFU/g
Log
CFU/g
221
46
27
1
228
34
0
2
31225
4.49
Rep 1
Dilution on
Plate
'Obsidian' Mid Harvest 07/20/11
Rep 2
Plate
1
'Triple Crown' Early-Harvest 08/23/11
Rep 2
Plate
1
Plate
2
CFU/g
Log
CFU/g
Plate
1
Plate 2
CFU/g
Log
CFU/g
38
2
1
0
28
7
0
0
3300
3.52
42
6
1
2
37
8
0
0
3950
3.60
Triple Crown' Mid/Late Harvest 08/30/11
Rep 1
Rep 2
Dilution on
Plate
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
CFU/g
Log
CFU/g
Plate
1
Plate 2
CFU/g
Log
CFU/g
TNTC
56
13
2
TNTC
27
7
3
41500
4.62
243
20
2
0
206
14
1
0
22450
4.35
67
Table I.2 Yeasts and Molds Raw Data, Fresh Samples
'Obsidian' Mid-Harvest 07/20/11
Rep 2
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
TNTC
107
13
TNTC
29
5
6800
3.83
TNTC
300
29
TNTC
550
40
34500
4.53
Obsidian' Late-Harvest 07/29/11
Rep 2
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
TNTC
257
63
TNTC
231
74
53367
4.73
TNTC
239
50
TNTC
270
49
40967
4.61
'Triple Crown' Early-Harvest 08/23/11
Rep 2
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
141
16
1
97
24
2
1190
3.01
117
29
1
100
33
10
2093
3.32
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
Triple Crown' Mid/Late Harvest 08/30/11
Rep 2
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
209
68
15
170
77
14
4572
3.66
185
102
3
210
85
9
5663
3.75
68
Table I.3 Coliforms Raw Data, Fresh Samples
'Obsidian' Mid-Harvest 07/20/11
Rep 2
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
0
0
0
0
0
0
<5
<0.70
11
0
0
14
0
0
125
2.10
'Obsidian' Late-Harvest 07/29/11
Rep 2
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
Dilution on
Plate
10^-1
10^-2
10^-3
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
0
0
0
0
0
0
<5
<0.70
0
0
0
0
0
0
<5
<0.70
Rep 1
'Triple Crown' Early-Harvest 08/23/11
Rep 2
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
4
0
0
1
0
0
25
1.40
0
0
0
0
0
0
<5
<0.70
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
'Triple Crown' Mid/Late-Harvest 08/30/11
Rep 2
Plate 1
Plate 2
CFU/g
Log CFU/g
Plate 1
Plate 2
CFU/g
Log CFU/g
0
0
0
0
0
0
<5
<0.70
0
0
0
0
0
0
<5
<0.70
69
Appendix II. Mechanical Harvester Raw Data and Photos, Chapter 2. Bold indicates value used
in calculation.
Table II.1 Aerobic Plate Count Raw Data, Harvester
Location 1
Pre-Harvest
Dilution on
Plate
10^-1
10^-2
10^-3
Dilution on
Plate
10^-1
10^-2
10^-3
Plate
1
Plate
2
CFU/swab
Log
CFU/swab
2.95 TNTC TNTC
4600
53
39
4
0
Location 2
Pre-Harvest
Post-Harvest
3.66
TNTC TNTC
6
12
2
1
Plate
1
Plate
2
216
2
10
227
31
11
Plate
1
Plate
2
CFU/swab
Post-Harvest
Log
CFU/swab
Plate
1
900
CFU/swab
2510
Log
CFU/swab
Plate
1
Plate
2
CFU/swab
3.40 TNTC
38
5
Location 3
350
34
3
3600
Pre-Harvest
Dilution on
Plate
10^-1
10^-2
10^-3
TNTC TNTC
11 TNTC
37
63
Plate
2
CFU/swab
50000
Log
CFU/swab
3.56
Post-Harvest
Log
CFU/swab
Plate
1
Plate
2
CFU/swab
4.70
41
2
0
52
4
1
Log
CFU/swab
Plate
1
Plate
2
CFU/swab
Log
CFU/swab
2.29
221
24
1
171
22
0
1960
3.29
465
Log
CFU/swab
2.67
Location 4
Pre-Harvest
Dilution on
Plate
10^-1
10^-2
10^-3
Plate
1
Plate
2
14
1
0
25
2
0
CFU/swab
195
Post-Harvest
Location 5
Pre-Harvest
Dilution on
Plate
10^-1
10^-2
10^-3
Plate
1
Plate
2
350 TNTC
43
41
21
3
CFU/swab
4200
Post-Harvest
Log
CFU/swab
Plate
1
Plate
2
3.62
400
47
2
350
44
20
CFU/swab
4500
Log
CFU/swab
3.66
Location 6
Pre-Harvest
Dilution on
Plate
Plate
1
Plate
2
CFU/swab
Post-Harvest
Log
CFU/swab
Plate
1
Plate
2
CFU/swab
Log
CFU/swab
70
10^-1
10^-2
10^-3
88
9
0
101
4
1
945
2.98
221
24
1
171
22
0
1960
3.29
Location 7
Pre-Harvest
Dilution on
Plate
10^-1
10^-2
10^-3
Plate
1
Plate
2
9
0
0
31
7
0
CFU/swab
200
Post-Harvest
Log
CFU/swab
Plate
1
Plate
2
2.30
27
3
2
23
2
0
Log
CFU/swab
Plate
1
Plate
2
1
111
12
7
88
13
13
CFU/swab
250
Log
CFU/swab
2.40
Location 8
Pre-Harvest
Dilution on
Plate
10^-1
10^-2
10^-3
Plate
1
Plate
2
0
0
0
2
0
0
CFU/swab
Figure II.1 Harvester Location 1
10
Post-Harvest
CFU/swab
Figure II.2 Harvester Location 2
995
Log
CFU/swab
2.99
71
Figure II-3 Harvester Location 3
Figure II.5 Harvester Location 5
Figure II.4 Harvester Location 4 (beater bar)
Figure II.6 Harvester Location 6
72
Figure II.7 Harvester Location 7
Figure II.8 Harvester Location 8
Appendix III. ‘Himalaya’ Raw Data, Chapter 2. Bold indicates value used in calculation.
Detection limit calculated as: <1 colony/plate at the lowest dilution with 0.1 ml on each of 2
plates  1/0.2 = 5 CFU/g or 0.70 Log CFU/g; 1/0.02 = 50 CFU/g or 1.70 Log CFU/g for
homogenized samples.
Table III.1 E. coli O157:H7 on fresh 'Himalaya' Blackberries, Raw Data
Rep 1
Berry Weight
1.4
Inoculum
5.74
g
Rep 2
1.6
Log
CFU/g
g
5.69
Log CFU/g
Rinse Solution
Dilution on Plate
10^-1
10^-2
10^-3
Homogenized 1:10
Dilution on Plate
Plate
1
Plate
2
Survivors
Log
CFU/g
Plate
1
Plate
2
Survivors
Log
CFU/g
0
0
0
0
0
0
<5
<0.70
0
0
0
0
0
0
<5
<0.70
73
10^-2
0
0
10^-3
0
0
<50
<1.70
0
0
0
0
<50
<1.70
Table III.2 Salmonella Typhimurium on fresh 'Himalaya' Blackberries, Raw Data
Rep 1
Rep 2
Berry Weight
1.3
Inoculum
5.80
g
1.1
Log
CFU/g
5.87
g
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
10^-3
Survivors
Log
CFU/g
<0.70
Plate
1
0
Plate 2
Survivors
0
<5
Log
CFU/g
<0.70
0
4
0
0
0
0
0
10^-2
0
0
0
0
<50
<1.70
10^-3
0
0
0
0
<5
Homogenized
1:10
Dilution on Plate
<50
<1.70
Table III.3 L. monocytogenes on fresh 'Himalaya' Blackberries, Raw Data
Rep 1
Berry Weight
1
Inoculum
6.25
g
Rep 2
0.6
Log
CFU/g
g
6.47
Log CFU/g
Plate
1
0
Plate
2
0
Survivors
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
Survivors
10^-2
0
0
0
0
10^-3
0
0
0
0
10^-2
0
0
0
0
10^-3
0
0
0
0
<5
Log
CFU/g
<0.70
<5
Log
CFU/g
<0.70
<50
<1.70
Homogenized 1:10
Dilution on Plate
<50
<1.70
74
Table III.4 S. aureus on fresh 'Himalaya' Blackberries, Raw Data
Rep 1
Berry Weight
1.2
Inoculum
5.33
Rep 2
g
1.7
Log
CFU/g
g
5.18
Log CFU/g
Plate
1
0
Plate
2
0
Survivors
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
Survivors
Log
CFU/g
<0.70
10^-2
0
0
0
0
10^-3
0
0
0
0
10^-2
2
2
1
0
10^-3
0
0
0
0
<5
<5
Log
CFU/g
<0.70
50
1.70
Homogenized 1:10
Dilution on Plate
200
2.30
Appendix IV. ‘Triple Crown’ Raw Data, Chapter 2. Bold indicates value used in calculation.
Detection limit calculated as: <1 colony/plate at the lowest dilution with 0.1 ml on each of 2
plates  1/0.2 = 5 CFU/g or 0.70 Log CFU/g; 1/0.02 = 50 CFU/g or 1.70 Log CFU/g for
homogenized samples.
Table IV.1 E. coli O157:H7 on Frozen 'Triple Crown' Blackberries, Raw Data
Rep 1
Rep 2
Berry Weight
8.7
Inoculum
4.40
g
6.9
Log
CFU/g
4.50
g
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
10^-3
0
10^-2
0
0
10^-3
0
0
Survivors
Log
CFU/g
<0.70
Plate
1
0
Plate 2
Survivors
0
<5
Log
CFU/g
<0.70
0
0
0
0
0
0
0
0
<50
<1.70
0
0
<5
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 3
Berry Weight
Inoculum
Rinse Solution
Rep 4
7.7
4.45
g
Log
CFU/g
8.3
4.42
g
Log
CFU/g
75
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
10^-3
0
10^-2
0
0
10^-3
0
0
Survivors
Log
CFU/g
<0.70
Plate
1
0
Plate 2
Survivors
0
<5
Log
CFU/g
<0.70
0
0
0
0
0
0
0
0
<50
<1.70
0
0
<5
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 5
Berry Weight
Rep 6
10
Inoculum
g
4.34
9.6
Log
CFU/g
4.36
g
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
10^-3
Survivors
Log
CFU/g
<0.70
Plate
1
0
Plate 2
Survivors
0
<5
Log
CFU/g
<0.70
0
0
0
0
0
0
0
10^-2
0
0
0
0
<50
<1.70
10^-3
0
0
0
0
<5
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 7
Berry Weight
10.6
g
Inoculum
4.31
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
0
10^-3
0
0
10^-2
0
0
10^-3
0
0
Survivors
<5
Log
CFU/g
<0.70
<50
<1.70
Homogenized
1:10
Dilution on Plate
76
Table IV.2 Salmonella Typhimurium on Frozen 'Triple Crown' Blackberries, Raw Data
Rep 1 at 3 months
Rep 2
Berry Weight
9.5
Inoculum
3.95
g
10.5
g
Log
CFU/g
3.90
Log
CFU/g
Plate
1
0
Plate
2
0
Survivors
Rinse Solution
Dilution on Plate
10^-1
Plate
1
1
Plate
2
1
Survivors
Log
CFU/g
1.00
10^-2
0
1
0
0
10^-3
0
0
0
0
10^-2
0
0
0
0
10^-3
0
0
0
0
10
<5
Log
CFU/g
<0.70
<50
<1.70
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 3
Berry Weight
Rep 4
7.7
Inoculum
4.04
g
9.3
Log
CFU/g
g
3.96
Log
CFU/g
Plate
1
0
Plate
2
0
Survivors
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
Survivors
Log
CFU/g
<0.70
10^-2
0
0
0
0
10^-3
0
0
0
0
10^-2
0
0
0
0
10^-3
0
0
0
0
<5
<5
Log
CFU/g
<0.70
<50
<1.70
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 5
Rep 6
Berry Weight
10.3
g
11.3
g
Inoculum
3.91
Log
CFU/g
3.87
Log
CFU/g
Plate
1
0
Plate
2
0
Survivors
Rinse Solution
Dilution on Plate
10^-1
Plate
1
1
Plate
2
0
10^-2
0
0
0
0
10^-3
0
0
0
0
Homogenized
1:10
Dilution on Plate
Survivors
5
Log
CFU/g
0.70
<5
Log
CFU/g
<0.70
77
10^-2
0
0
10^-3
0
0
<50
<1.70
0
0
0
1
<50
<1.70
Rep 7
Berry Weight
8.4
Inoculum
4.00
g
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
0
10^-3
0
0
10^-2
0
0
10^-3
0
0
Survivors
<5
Log
CFU/g
<0.70
<50
<1.70
Homogenized
1:10
Dilution on Plate
Table IV.3 L. monocytogenes on Frozen 'Triple Crown' Blackberries, Raw Data
Rep 1
Rep 2
Berry Weight
9.6
Inoculum
4.32
g
7.7
Log
CFU/g
g
4.42
Log
CFU/g
Plate
1
1
Plate
2
1
Survivors
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
Survivors
Log
CFU/g
<0.70
10^-2
0
0
0
0
10^-3
0
0
0
0
10^-2
0
1
2
0
10^-3
0
0
0
0
<5
10
Log
CFU/g
1
100
2
Homogenized
1:10
Dilution on Plate
50
1.70
Rep 3
Rep 4
Berry Weight
11.5
g
9.8
Inoculum
4.24
Log
CFU/g
4.31
g
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
0
Survivors
<5
Log
CFU/g
<0.70
Plate
1
0
Plate 2
Survivors
0
<5
0
0
Log
CFU/g
<0.70
78
10^-3
0
0
10^-2
0
0
10^-3
0
0
0
0
0
0
0
0
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 5
Berry Weight
<1.70
Rep 6
5.7
Inoculum
<50
4.55
g
11.7
g
Log
CFU/g
4.24
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
10^-3
Survivors
Log
CFU/g
<0.70
Plate
1
0
Plate 2
Survivors
0
<5
Log
CFU/g
<0.70
0
0
0
0
0
0
0
10^-2
0
0
0
1
50
1.70
10^-3
0
0
0
0
<5
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 7
Berry Weight
10.8
g
Inoculum
4.27
Log
CFU/g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
0
10^-3
0
0
10^-2
0
0
10^-3
0
0
Survivor
s
<5
Log
CFU/g
<0.70
<50
<1.70
Homogenized
1:10
Dilution on Plate
Table IV.4 S. aureus on Frozen 'Triple Crown' Blackberries, Raw Data
Rep 1
Berry Weight
Inoculum
Rinse Solution
8.7
3.59
g
Log
CFU/g
Rep 2
7.8
3.63
g
Log
CFU/g
79
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
10^-3
0
10^-2
2
1
10^-3
0
0
Survivors
Log
CFU/g
<0.70
Plate
1
1
Plate
2
0
0
0
0
0
0
0
0
0
0
0
<5
Survivors
5
Log
CFU/g
0.70
<50
<1.70
Homogenized
1:10
Dilution on Plate
150
2.18
Rep 3
Berry Weight
Rep 4
8.9
Inoculum
3.58
g
9.3
Log
CFU/g
g
3.56
Log
CFU/g
Plate
1
1
Plate
2
1
Survivors
Rinse Solution
Dilution on Plate
10^-1
Plate
1
1
Plate
2
0
Survivors
Log
CFU/g
0.70
10^-2
0
0
0
0
10^-3
0
0
0
0
10^-2
0
0
1
0
10^-3
0
0
0
0
5
10
Log
CFU/g
1.00
50
1.70
3.54
Log
CFU/g
Plate
1
0
Plate
2
0
Survivors
<5
Log
CFU/g
0.70
100
2.00
Homogenized
1:10
Dilution on Plate
<50
<1.70
Rep 5
Berry Weight
Rep 6
8.5
Inoculum
3.60
g
9.6
Log
CFU/g
g
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
Survivors
Log
CFU/g
0.70
10^-2
0
0
0
0
10^-3
0
0
0
0
10^-2
5
12
2
0
10^-3
0
0
0
0
<5
Homogenized
1:10
Dilution on Plate
850
2.93
Rep 7
Berry Weight
Inoculum
7.9
3.63
g
Log
CFU/g
80
Rinse Solution
Dilution on Plate
10^-1
Plate
1
0
Plate
2
0
10^-2
0
0
10^-3
0
0
10^-2
1
0
10^-3
0
0
Survivors
<5
Log
CFU/g
<0.70
50
1.70
Homogenized
1:10
Dilution on Plate
Appendix V. Juice Raw Data, Chapter 3. Bold indicates value used in calculation. Detection limit
calculated as: <1 colony/plate at the lowest dilution with 0.1 ml on each of 2 plates  1/0.2 = 5
CFU/ml or 0.70 Log CFU/ml.
Table V.1 E. coli O157:H7 in 'Marion' Juice, Raw Data
Rep 1 12/14/11
Time
Dilution on Plate
Plate 1
Plate 2
6h
10^-1
TNTC
TNTC
10^-2
TNTC
TNTC
10^-3
112
127
10^-4
25
28
10^-5
0
4
10^-6
1
0
12h
10^-1
TNTC
TNTC
10^-2
194
215
10^-3
56
46
10^-4
6
8
10^-5
0
0
10^-6
0
0
24h
10^-1
TNTC
TNTC
10^-2
66
98
10^-3
10
3
36h
10^-1
91
82
10^-2
0
2
48h
10^-1
0
0
60h
10^-1
--72h
10^-1
--84h
10^-1
--Rep 2 8/22/12
Survivors
192250
Log CFU/ml
5.28
35725
4.55
8200
3.91
865
2.94
<5
<0.70
81
Time
6h
12h
24h
36h
48h
60h
72h
84h
Dilution on Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
-10^-1
10^-2
10^-3
10^-4
10^-5
-10^-6
-10^-1
10^-2
10^-3
10^-1
10^-2
10^-1
10^-1
10^-1
10^-1
Plate 1
TNTC
TNTC
223
44
7
Plate 2
TNTC
TNTC
191
34
2
Survivors
298500
Log CFU/ml
5.474
TNTC
TNTC
105
14
108500
5.04
TNTC
78
6
24
0
0
1
0
0
7000
3.85
230
2.36
20
10
5
<5
1.30
1.00
0.70
<0.70
Survivors
84500
Log CFU/ml
4.93
14500
4.16
1570
3.20
-TNTC
TNTC
112
19
--TNTC
62
13
22
1
4
1
1
0
Table V.2 E. coli O157:H7 in 'Black Diamond' Juice, Raw Data
Rep 1 12/14/11
Time
Dilution on Plate
Plate 1
Plate 2
6h
10^-1
TNTC
TNTC
10^-2
300
300
10^-3
85
84
10^-4
15
15
10^-5
2
1
10^-6
0
0
12h
10^-1
TNTC
TNTC
10^-2
156
134
10^-3
23
10
10^-4
5
1
10^-5
2
0
10^-6
0
0
24h
10^-1
125
189
10^-2
18
7
82
36h
48h
60h
72h
84h
Time
6h
12h
24h
36h
48h
60h
72h
84h
10^-3
10^-1
10^-2
106-3
10^-1
10^-1
10^-1
10^-1
0
1
0
--
Dilution on Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
-10^-1
10^-2
10^-3
10^-4
10^-5
-10^-6
-10^-1
10^-2
10^-3
10^-1
10^-2
10^-3
10^-1
10^-1
10^-1
10^-1
1
3
0
20
1.30
5
5
10
<5
0.70
0.70
1.00
<0.70
Survivors
327250
Log CFU/ml
5.51
58000
4.76
4800
3.68
275
2.44
30
15
10
<5
1.48
1.18
1.00
<0.70
-1
1
2
0
0
0
0
0
Rep 2 8/22/12
Plate 1
Plate 2
TNTC
TNTC
TNTC
TNTC
155
194
45
51
3
3
-TNTC
TNTC
TNTC
TNTC
54
62
10
12
--TNTC
TNTC
54
42
8
8
28
27
1
1
1
0
5
1
3
0
2
0
0
0
Table V.3 Salmonella Typhimurium in 'Marion' Juice, Raw Data
Rep 1 12/14/11
Time
Dilution on Plate
Plate 1
Plate 2
Survivors
6h
10^-1
TNTC
TNTC
11750
10^-2
85
150
10^-3
18
21
10^-4
5
7
Log CFU/ml
4.07
83
12h
24h
36h
48h
60h
Time
6h
12h
24h
36h
48h
60h
10^-5
10^-6
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
10^-1
10^-2
10^-1
10^-1
10^-1
Dilution on Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
10^-1
10^-2
10^-1
10^-1
10^-1
--
------
--
0
0
0
0
219
131
13
6
1
0
0
0
0
0
0
0
12
13
2
0
3
2
1
2
0
0
Rep 2 08/14/12
Plate 1
Plate 2
57
58
2
3
1
0
0
0
--21
16
1
1
----1
2
-1
0
0
0
--
1750
3.24
125
2.10
25
15
<5
1.40
1.18
<0.70
Survivors
575
Log CFU/ml
2.76
185
2.27
15
1.18
5
<5
0.70
<0.70
Table V.4 Salmonella Typhimurium in 'Black Diamond' Juice, Raw Data
Rep 1 12/14/11
Time
Dilution on Plate
Plate 1
Plate 2
Survivors
6h
10^-1
TNTC
TNTC
7325
10^-2
142
151
10^-3
24
21
10^-4
4
2
Log CFU/ml
3.86
84
12h
24h
36h
48h
60h
72h
84h
96h
108h
Time
6h
12h
24h
36h
48h
60h
72h
84h
96h
108h
10^-5
10^-6
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
10^-1
10^-2
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
Dilution on Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
10^-1
10^-2
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
10^-1
---
------
-----
0
0
0
0
37
34
1
3
1
1
0
0
0
0
0
0
3
7
0
0
4
2
1
6
0
1
1
2
1
1
1
0
0
0
Rep 2 08/14/12
Plate 1
Plate 2
76
67
3
2
0
0
0
0
--26
31
0
0
----36
3
-3
1
2
1
0
0
-----
355
2.55
50
1.70
30
35
5
15
10
5
<5
1.48
1.54
0.70
1.18
1.00
0.70
<0.70
Survivors
715
Log CFU/ml
2.85
285
2.45
195
2.29
20
15
<5
1.30
1.18
<0.70
85
Table V.5 L. monocytogenes in 'Marion' Juice, Raw Data
Rep 1 03/03/12
Time
Dilution on Plate
Plate 1
Plate 2
3h
10^-1
--10^-2
--10^-3
--10^-4
--6h
10^-1
35
51
10^-2
1
0
10^-3
0
0
10^-4
0
0
10^-5
0
0
10^-6
0
0
12h
10^-1
0
0
10^-2
0
0
10^-3
0
0
10^-4
0
0
10^-5
0
0
Rep 2 09/05/12
Time
Dilution on Plate
Plate 1
Plate 2
3h
10^-1
2
5
10^-2
0
0
10^-3
0
0
10^-4
0
0
6h
10^-1
0
0
10^-2
0
0
10^-3
--10^-4
--10^-5
--10^-6
--12h
10^-1
--10^-2
--10^-3
--10^-4
--10^-5
---
Survivors
Log CFU/ml
430
2.63
<5
<0.70
Survivors
35
Log CFU/ml
1.54
<5
<0.70
86
Table V.6 L. monocytogenes in 'Black Diamond' Juice, Raw Data
Rep 1 03/03/12
Time
Dilution on Plate
Plate 1
Plate 2
Survivors
3h
10^-1
--10^-2
--10^-3
--10^-4
--6h
10^-1
10
16
130
10^-2
0
0
10^-3
0
0
10^-4
0
0
10^-5
0
0
10^-6
0
0
12h
10^-1
0
0
<5
10^-2
0
0
10^-3
0
0
10^-4
0
0
10^-5
0
0
Rep 2 09/05/12
Time
Dilution on Plate
Plate 1
Plate 2
Survivors
3h
10^-1
0
0
<5
10^-2
0
0
10^-3
0
0
10^-4
0
0
6h
10^-1
0
0
10^-2
--10^-3
--10^-4
--10^-5
--10^-6
--12h
10^-1
--10^-2
--10^-3
--10^-4
--10^-5
---
Log CFU/ml
2.11
<0.70
Log CFU/ml
<0.70
87
Table V.7 S. aureus in 'Marion' Juice, Raw Data
Rep 1 04/11/12
Time
Dilution on Plate
Plate 1
Plate 2
6h
10^-1
TNTC
TNTC
10^-2
290
230
10^-3
30
27
10^-4
--12h
10^-1
TNTC
TNTC
10^-2
109
139
10^-3
--10^-4
--24h
10^-1
208
206
10^-2
19
10
36h
10^-1
2
2
10^-2
0
0
48h
10^-1
0
0
Rep 2 09/18/12
Time
Dilution on Plate
Plate 1
Plate 2
6h
10^-1
80
101
10^-2
5
5
10^-3
0
0
10^-4
0
0
12h
10^-1
2
3
10^-2
0
0
10^-3
0
0
10^-4
0
0
24h
10^-1
0
0
10^-2
0
0
36h
10^-1
--10^-2
--48h
10^-1
---
Table V.8 S. aureus in 'Black Diamond' Juice, Raw Data
Rep 1 03/23/12
Time
Dilution on Plate
Plate 1
Plate 2
6h
10^-1
TNTC
TNTC
10^-2
126
71
10^-3
8
15
10^-4
12h
10^-1
TNTC
TNTC
Survivors
26667
Log CFU/ml
4.43
12400
4.09
2070
3.32
20
1.30
<5
<0.70
Survivors
905
Log CFU/ml
2.96
25
1.40
<5
<0.70
Survivors
9850
Log CFU/ml
3.99
8100
3.91
88
24h
36h
48h
60h
72h
84h
Time
6h
12h
24h
36h
48h
60h
72h
84h
10^-2
10^-3
10^-4
10^-1
10^-2
10^-3
10^-1
10^-2
10^-3
10^-1
10^-1
10^-1
10^-1
Dilution on Plate
10^-1
10^-2
10^-3
10^-4
10^-1
10^-2
10^-3
10^-4
10^-1
10^-2
10^-3
10^-1
10^-2
10^-3
10^-1
10^-1
10^-1
10^-1
105
7
--
----
---
57
5
-TNTC
TNTC
19
17
1
2
222
350
3
3
1
0
107
89
16
12
3
5
0
0
Rep 2 09/18/12
Plate 1
Plate 2
TNTC
TNTC
119
132
13
32
3
5
236
297
22
18
2
1
4
0
29
17
2
0
-1
0
--1
0
0
0
---
1800
3.26
2860
3.46
980
140
40
<5
2.99
2.15
1.60
<0.70
Survivors
19033
Log CFU/ml
4.28
2665
3.43
230
2.36
5
0.70
5
<5
0.70
<0.70
89
Appendix VI. Juice Variables Raw Data, Chapter 3. Bold indicates value used in calculation.
Detection limit calculated as: <1 colony/plate at the lowest dilution with 0.1 ml on each of 2
plates  1/0.2 = 5 CFU/ml or 0.70 Log CFU/ml.
Table VI.1 E. coli O157:H7 in 'Marion' Juice with added ethanol, Raw Data 08/22/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
191
80
7
TNTC
TNTC
169
43
12
397500
5.60
TNTC
TNTC
184
41
1
TNTC
TNTC
131
42
6
286250
5.46
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
TNTC TNTC
135
82
58
40
1
0
--12h
29925
4.48
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Dilution on
Plate
10^-1
Plate
1
38
1
0
0
0
36h
10^-2
10^-3
10^-4
10^-5
Dilution on
Plate
10^-1
Plate
1
10^-2
-----
10^-3
10^-4
10^-5
TNTC TNTC
Error Error
Error Error
Error Error
--24h
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
27
0
0
0
0
325
2.51
1
0
5
0.70
----Survivors
0
-----
Log
CFU/ml
Plate
2
Plate
2
0
6h
<5
Log
CFU/ml
<0.70
-----
90
Table VI.2 E. coli O157:H7 in 'Black Diamond' Juice with added ethanol, Raw Data 08/22/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
152
37
5
TNTC
TNTC
141
21
7
221000
5.34
TNTC
TNTC
77
10
2
TNTC
TNTC
95
9
1
86000
4.93
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
107
194
54
40
7
5
---
31025
4.49
TNTC TNTC
45
28
5
3
1
0
---
3650
3.56
Survivors
Log
CFU/ml
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
6h
12h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
24h
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
114
11
2
0
0
80
11
0
0
0
970
2.99
2
0
Plate
1
Plate
2
Survivors
Log
CFU/ml
0
<5
<0.70
----
10
1.00
----
36h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
0
----
----
Table VI.3 Salmonella Typhimurium in 'Marion' Juice with added ethanol, Raw Data 08/14/2012
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
169
3
0
0
0
192
10
0
0
0
1805
3.26
97
1
0
0
0
90
0
0
0
0
935
2.97
91
4h
Dilution on
Plate
10^-1
6h
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
2
0
0
0
0
3
0
0
0
0
25
1.40
0
0
0
0
0
0
0
0
0
0
<5
<0.70
10^-2
10^-3
10^-4
10^-5
Table VI.4 Salmonella Typhimurium in 'Black Diamond' Juice with added ethanol , Raw Data
08/14/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log CFU/ml
Plate
1
Plate
2
Survivors
Log CFU/ml
15
1
0
0
0
12
0
0
0
0
135
2.13
1
0
0
0
0
5
0
0
0
0
30
1.48
Plate
1
Plate
2
Survivors
Log CFU/ml
Plate
1
Plate
2
Survivors
Log CFU/ml
1
0
0
0
0
0
0
0
0
0
5
0.70
0
0
0
0
0
0
0
0
0
0
<5
0.70
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
6h
Table VI.5 L. monocytogenes in 'Marion' Juice with added ethanol, Raw Data 09/05/12
0.5h
1h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
41
1
1
0
TNTC
58
7
0
0
4950
3.69
20
0
0
0
0
25
0
0
0
0
22.5
1.35
Plate
1
Plate
2
Survivors
Log
CFU/ml
2h
Dilution on
Plate
92
10^-1
10^-2
10^-3
10^-4
10^-5
0
0
0
0
0
0
0
0
0
0
<5
<0.70
Table VI.6 L. monocytogenes in 'Black Diamond' Juice with added ethanol , Raw Data 09/05/12
0.5h
1h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
22
0
0
0
0
29
0
0
0
0
25.5
1.41
0
0
0
0
0
0
0
0
0
0
<5
<0.70
Table VI.7 S. aureus in 'Marion' Juice with added ethanol , Raw Data 09/18/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
95
16
0
TNTC
TNTC
73
15
1
84000
4.92
TNTC
193
58
9
0
TNTC
203
39
11
0
34150
4.53
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
101
72
12
3
0
0
---
8650
3.94
TNTC TNTC
50
61
5
2
0
0
---
5550
3.74
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
6h
12h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
24h
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
16
2
0
0
6
0
0
0
110
2.04
0
0
<5
<0.70
----
----
93
10^-5
--
--
--
--
Table VI.8 S. aureus in 'Black Diamond' Juice with added ethanol , Raw Data 09/18/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
143
35
2
TNTC
TNTC
108
42
0
255250
5.41
TNTC
243
92
20
0
TNTC
TNTC
102
20
0
72767
4.86
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
101
65
13
10
4
0
---
8300
3.92
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
6h
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
160
171
10
3
0
1
---
16550
4.22
Plate
1
12h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
24h
Plate
1
Plate
2
Survivors
Log
CFU/ml
100
13
0
0
79
8
0
0
895
2.95
--
10^-2
10^-3
10^-4
10^-5
-----
-Plate
1
Plate
2
Survivors
Log
CFU/ml
0
0
<5
<0.70
-----
6
----
36h
Dilution on
Plate
10^-1
Plate
1
----
Plate
2
Survivors
Log
CFU/ml
4
50
1.70
94
Appendix VII. Wine Raw Data, Chapter 3. Bold indicates value used in calculation. Detection
limit calculated as: <1 colony/plate at the lowest dilution with 0.1 ml on each of 2 plates 
1/0.2 = 5 CFU/ml or 0.70 Log CFU/ml.
Table VII.1 E. coli O157:H7 in 'Marion' Wine, Raw Data
Rep 1 12/09/11
10m
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
194
38
3
1
0
TNTC
190
43
2
0
0
29850
4.47
91
3
0
0
0
0
112
0
0
0
0
0
1015
3.01
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
TNTC
222
-----
22200
4.35
Rep 1 12/09/11
40m
Dilution on
Plate
10^-1
Plate
1
Plate
2
Survivors
Log
CFU/ml
0
0
0
0
0
0
0
0
0
0
0
0
<5
<0.70
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
TNTC
87
TNTC
TNTC
TNTC
68
775000
5.89
Plate
1
Plate
2
Survivors
Log
CFU/ml
36
375
2.57
10^-2
10^-3
10^-4
10^-5
10^-6
Rep 2 07/30/12
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10m
Rep 2 07/30/12
10^-3
10^-4
20m
40m
Dilution on
Plate
10^-1
10^-2
20m
39
----
----
60m
Plate
1
0
----
----
Plate
2
Survivors
Log
CFU/ml
0
<5
<0.70
95
Table VII.2 E. coli O157:H7 in 'Black Diamond' Wine, Raw Data
Rep 1
10m
12/09/11
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
Rep 1
12/09/11
Dilution on
Plate
10^-1
Plate 1
Plate 2
Survivors
Log
CFU/ml
Plate 1
Plate 2
Survivors
Log
CFU/ml
TNTC
TNTC
197
67
10
1
TNTC
TNTC
182
74
12
3
447250
5.65
240
113
17
0
0
0
232
104
14
0
0
0
6605
3.82
40m
Plate 1
Plate 2
Survivors
Log
CFU/ml
0
0
0
0
0
0
0
0
0
0
0
0
<5
<0.70
10^-2
10^-3
10^-4
10^-5
10^-6
Rep 2
07/30/12
Dilution on
Plate
10^-1
10m
Plate 2
Survivors
Log
CFU/ml
Plate 1
Plate 2
Survivors
Log
CFU/ml
30
5
1
0
42
8
1
0
360
2.56
0
0
0
0
0
0
0
0
<5
<0.70
10^-3
10^-4
10^-2
10^-3
10^-4
20m
Plate 1
10^-2
Rep 2
07/30/12
Dilution on
Plate
10^-1
20m
40m
Plate
1
-----
Plate
2
-----
--
--
96
Table VII.3 Salmonella Typhimurium in 'Marion' Wine, Raw Data
Rep 1
10m
12/07/11
Dilution on
Plate
10^-1
Plate 1
Plate 2
Survivors
248
23
1
0
0
0
262
35
0
0
0
0
2760
10^-2
10^-3
10^-4
10^-5
10^-6
Rep 1
12/07/11
Dilution on
Plate
10^-1
Plate 2
Survivors
Log CFU/ml
0
0
0
0
0
0
0
0
0
0
0
0
<5
<0.70
10^-4
10^-5
10^-6
10^-3
Rep 2
07/22/12
Dilution on
Plate
10^-1
10^-2
10^-3
Plate
2
3
0
0
0
0
0
1
0
0
0
0
0
3.44
Plate 1
10^-3
10^-2
Plate
1
10^-2
10^-3
Log
CFU/ml
20
1.30
Plate
2
Survivors
Log
CFU/ml
52
480
2.68
10m
20m
Plate 1
Plate 2
Survivors
Log CFU/ml
TNTC
TNTC
51
TNTC
TNTC
37
44000
4.64
Plate
1
44
---
---
40m
Plate 1
Plate 2
Survivors
Log CFU/ml
0
0
<5
<0.70
---
---
Table VII. 4 Salmonella Typhimurium in 'Black Diamond' Wine, Raw Data
Rep 1
10m
12/07/11
Dilution on
Plate
10^-1
Survivors
40m
10^-2
Rep 2
07/22/12
Dilution on
Plate
10^-1
Log CFU/ml
20m
20m
Plate 1
Plate 2
Survivors
Log CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
204
TNTC
TNTC
189
365750
5.56
TNTC
263
51
TNTC
205
60
43833
4.64
97
10^-4
65
2
0
10^-5
10^-6
Rep 1
12/07/11
Dilution on
Plate
10^-1
Plate 1
Plate 2
Survivors
Log CFU/ml
0
0
0
0
0
0
0
0
0
0
0
0
<5
<0.70
10^-3
10^-4
10^-5
10^-6
10^-2
10^-3
Rep 2
07/22/12
Dilution on
Plate
10^-1
10^-2
10^-3
7
1
0
7
2
0
40m
10^-2
Rep 2
07/22/12
Dilution on
Plate
10^-1
42
10
0
10m
20m
Plate 1
Plate 2
Survivors
Log CFU/ml
TNTC
173
23
TNTC
151
23
16200
4.21
Plate 1
Plate 2
1
---
0
10^-2
10^-3
10^-4
10^-5
10^-6
0.70
40m
Plate 1
Plate 2
Survivors
0
<5
0
---
Log CFU/ml
<0.70
---
20m
Plate 1
Plate 2
Survivors
Log
CFU/ml
Plate
1
Plate 2
Survivors
Log CFU/ml
TNTC
TNTC
TNTC
159
10
4
TNTC
TNTC
TNTC
176
17
1
1675000
6.22
TNTC
TNTC
294
51
3
0
TNTC
TNTC
286
33
2
0
420000
5.62
Plate 1
Plate 2
Survivors
Log
CFU/ml
Plate
1
Plate 2
Survivors
Log CFU/ml
54
47
505
2.70
0
0
<5
<0.70
Rep 1
Dilution on
Plate
10^-1
5
Log
CFU/ml
---
Table VII.5 L. monocytogenes in 'Marion' Wine, Raw Data
Rep 1
10m
02/08/12
Dilution on
Plate
10^-1
Survivors
40m
60m
98
10^-2
4
0
0
0
0
10^-3
10^-4
10^-5
10^-6
Rep 2
08/01/12
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
3
0
0
0
0
10^-2
10^-3
10^-4
10^-5
20m
Plate 1
Plate 2
Survivors
Log
CFU/ml
Plate
1
Plate 2
Survivors
Log
CFU/ml
TNTC
TNTC
TNTC
172
20
TNTC
TNTC
TNTC
201
30
2243333
6.35
TNTC
TNTC
TNTC
93
10
TNTC
TNTC
TNTC
127
8
1100000
6.04
Plate 1
Plate 2
Survivors
Log
CFU/ml
Plate 1
Plate 2
Survivors
Log
CFU/ml
TNTC TNTC
246
320
-------
28300
4.45
0
10
1.00
40m
Rep 2
60m
Plate 2
Survivors
Log
CFU/ml
0
0
<5
<0.70
10^-2
-----
10^-5
-----
80m
Plate 1
10^-4
2
-----
Dilution on
Plate
10^-1
10^-3
0
0
0
0
0
10m
Rep 2
Dilution on
Plate
10^-1
0
0
0
0
0
-----
Table VII.6 L. monocytogenes in 'Black Diamond' Wine, Raw Data
Rep 1
10m
02/16/12
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
20m
Plate 1
Plate 2
Survivors
Log
CFU/ml
Plate 1
Plate 2
Survivors
Log CFU/ml
TNTC
TNTC
TNTC
120
15
1
TNTC
TNTC
TNTC
114
11
0
1170000
6.07
TNTC
TNTC
57
11
0
0
TNTC
TNTC
43
16
1
0
50000
4.70
99
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
Rep 2
08/01/12
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
40m
Plate 1
Plate 2
Survivors
Log
CFU/ml
0
0
0
0
0
0
0
0
0
0
0
0
<5
<0.70
10m
20m
Plate 1
Plate 2
Survivors
Log
CFU/ml
Plate 1
Plate 2
Survivors
Log CFU/ml
104
73
7
3
0
181
102
19
4
0
5088
3.71
0
0
0
0
0
0
0
0
0
0
<5
<0.70
Table VII.7 S. aureus in 'Marion' Wine, Raw Data
Rep 1
10m
02/14/12
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
Plate 1
Plate 2
Survivors
Log CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
150
36
7
0
0
TNTC
221
35
15
0
0
27025
4.43
TNTC
94
32
17
0
0
TNTC
176
37
2
0
0
24000
4.38
Plate 1
Plate 2
Survivors
Log CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
162
26
7
0
0
0
185
25
2
0
1
0
2143
3.33
4
3
0
0
0
0
3
1
0
0
0
0
35
1.54
Rep 1
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-6
40m
Rep 1
Dilution on
Plate
20m
60m
80m
Plate 1
Plate 2
Survivors
Log CFU/ml
100
10^-1
0
0
0
0
0
0
10^-2
10^-3
10^-4
10^-5
10^-6
Rep 2
07/22/12
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
0
0
0
0
0
0
Plate 2
Survivors
Log CFU/ml
TNTC
TNTC
240
65
3
TNTC
TNTC
180
40
3
367500
5.57
Plate 1
Plate 2
Survivors
Log CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
6
4
0
13
3
1
95
1.98
0
0
<5
<0.70
---
Log
CFU/ml
TNTC TNTC
TNTC TNTC
178
231
27
31
---
247250
5.39
-----
80m
Plate 1
Plate 2
Survivors
Log CFU/ml
0
0
<5
<0.70
10^-2
-----
10^-5
Survivors
-----
Dilution on
Plate
10^-1
10^-4
Plate
2
60m
---
Rep 2
10^-3
Plate
1
40m
10^-3
10^-5
20m
Plate 1
10^-2
10^-4
0.70
10m
Rep 2
Dilution on
Plate
10^-1
<5
-----
Table VII.8 S. aureus in 'Black Diamond' Wine, Raw Data
Rep 1
10m
02/23/12
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
20m
Plate
1
Plate 2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
159
24
2
TNTC
TNTC
99
22
1
129000
5.11
TNTC
TNTC
122
23
0
TNTC
TNTC
101
7
0
11500
5.05
101
10^-6
0
0
Plate
1
5876.66
7
Survivors
375
51
6
2
0
0
311
33
14
0
0
0
Rep 1
10^-2
10^-3
10^-4
10^-5
10^-6
Rep 1
Plate
1
Plate 2
0
0
0
0
0
0
0
0
0
0
0
0
10^-2
10^-3
10^-4
10^-5
10^-6
10^-3
10^-4
10^-5
10^-4
4200
3.77
1
0
0
0
0
0
2
0
0
0
0
0
15
1.18
Survivors
Log
CFU/ml
<5
<0.70
20m
Log
CFU/ml
TNTC
TNTC
161
21
3
TNTC
TNTC
168
16
4
164500
5.22
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
181
168
23
31
1
3
---
11497
4.06
40m
Plate
1
Plate 2
6
0
0
---
60m
Survivors
2
1
0
40
Log
CFU/ml
----
Plate
1
Plate 2
Survivors
Log
CFU/ml
0
0
<5
<0.70
Plate
2
0
-----
---
----
Plate
1
1.60
80m
Dilution on
Plate
10^-1
10^-3
Log
CFU/ml
Survivors
Rep 2
10^-2
Survivors
60m
Plate 2
10^-3
10^-5
Plate
2
Plate
1
10^-2
10^-4
Plate
1
10m
Rep 2
Dilution on
Plate
10^-1
Log
CFU/ml
80m
Dilution on
Plate
10^-1
10^-2
0
40m
Dilution on
Plate
10^-1
Rep 2
07/22/12
Dilution on
Plate
10^-1
0
Survivors
0
-----
<5
Log
CFU/ml
<0.70
102
10^-5
--
--
Appendix VIII. Wine Variable Raw Data, Chapter 3. Bold indicates value used in calculation.
Detection limit calculated as: <1 colony/plate at the lowest dilution with 0.1 ml on each of 2
plates  1/0.2 = 5 CFU/ml or 0.70 Log CFU/ml.
Table VIII.1 E. coli O157:H7 in pH adjusted 'Marion' Wine, Raw Data 07/30/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
TNTC TNTC
184 TNTC
88
58
5
19
54800
5.74
TNTC
TNTC
TNTC
56
6
TNTC
TNTC
TNTC
46
10
510000
5.70
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
6h
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
153
75
10
TNTC
TNTC
221
79
10
478500
5.68
TNTC
TNTC
100
49
3
TNTC
TNTC
188
51
3
322000
5.51
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
45
18
3
0
TNTC
88
3
2
0
6650
3.82
19
0
0
34
0
0
265
2.42
Plate
1
Plate
2
Survivors
Log
CFU/ml
0
0
0
0
<5
<0.70
12h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
24h
--36h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
----
----
---
103
Table VIII.2 E. coli O157:H7 in pH adjusted 'Black Diamond' Wine, Raw Data 07/30/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
115500
0
6.06
TNTC
TNTC
665000
5.82
TNTC
TNTC
134
15
TNTC
TNTC
97
16
TNTC
TNTC
74
2
TNTC
TNTC
59
11
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
TNTC
78
10
TNTC
TNTC
223
72
6
574333
5.76
TNTC
TNTC
122
38
3
TNTC
TNTC
259
40
2
300667
5.48
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
6h
12h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
24h
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
46
10
0
TNTC
131
41
13
0
33367
4.52
Plate
1
Plate
2
Survivors
Log
CFU/ml
10
0
0
54
0
0
320
2.51
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
59
35
10
5
-----
4700
3.67
36h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
---
48h
Plate
1
Survivors
Log
CFU/ml
0
<5
<0.70
0
-----
---
Plate
2
-----
Table VIII.3 Salmonella Typhimurium in pH adjusted 'Marion' Wine, Raw Data 07/22/12
6h
Dilution on
Plate
10^-1
10^-2
10^-3
Plate 1
Plate 2
Survivors
Log CFU/ml
0
0
0
0
0
0
<5
<0.70
104
Table VIII.4 Salmonella Typhimurium in pH adjusted 'Black Diamond' Wine, Raw Data 07/22/12
6h
12h
Dilution on
Plate
10^-1
10^-2
10^-3
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
8
0
0
8
0
0
80
1.90
9
3
60
1.78
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
1
0
5
0.70
0
0
<5
<0.70
---
---
24h
Dilution on
Plate
10^-1
10^-2
10^-3
---
36h
---
---
---
Table VIII.5 L. monocytogenes in pH adjusted 'Marion' Wine, Raw Data 08/01/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
127500
0
6.11
TNTC
TNTC
463000
5.67
TNTC
TNTC
156
8
TNTC
TNTC
99
7
TNTC
TNTC
55
4
TNTC
189
65
6
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
6h
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
223
16
1
TNTC
TNTC
112
20
0
167500
5.22
TNTC
TNTC
78
16
0
TNTC
TNTC
67
7
0
72500
4.86
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
179000
5.25
TNTC
TNTC
31766.6
7
4.50
TNTC
170
26
TNTC
188
8
12h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
24h
TNTC
123
56
27
---
105
10^-5
0
0
--
--
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
105
81
12
12
-----
9300
3.97
39
16
32
24
355
2.55
36h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
48h
----
----
Table VIII.6 L. monocytogenes in pH adjusted 'Black Diamond' Wine, 08/01/12
1h
2h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
TNTC
TNTC
147500
0
6.17
TNTC
TNTC
TNTC
TNTC
163
2
TNTC
TNTC
132
8
TNTC
325
63
3
TNTC
TNTC
25
2
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
174
25
1
TNTC
TNTC
201
8
1
208333
5.32
TNTC
TNTC
140
27
0
TNTC
TNTC
162
1
0
190667
5.28
4h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
10^-2
10^-3
10^-4
10^-5
10^-2
10^-3
5.64
24h
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
TNTC
227
26
2
TNTC
TNTC
111
15
0
199333
5.30
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
TNTC TNTC
92
21
-----
56500
4.75
36h
Dilution on
Plate
10^-1
440000
Log
CFU/ml
6h
12h
Dilution on
Plate
10^-1
Survivors
48h
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC
150
38
TNTC
203
7
24433
4.39
Plate
1
Plate
2
Survivors
Log
CFU/ml
TNTC TNTC
142
181
---
16150
4.21
106
10^-4
10^-5
---
---
---
---
Table VIII.7 S. aureus in pH adjusted 'Marion' Wine, Raw Data 07/22/12
6h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
12h
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
253
18
0
0
0
180
41
0
0
0
2950
3.47
61
2
0
0
0
21
1
0
0
0
410
2.61
24h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
Plate
1
Plate
2
1
0
0
0
---
36h
Survivors
5
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
0.70
0
0
<5
<0.70
Survivors
Log
CFU/ml
----
---
----
10^-5
Table VIII.8 S. aureus in pH adjusted 'Black Diamond' Wine, Raw Data 07/22/12
6h
12h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
Plate
1
Plate
2
116
5
2
0
0
136
24
1
0
0
Survivors
1260
Log
CFU/ml
Plate
1
Plate
2
3.10
15
0
0
0
0
19
0
0
0
0
24h
Dilution on
Plate
10^-1
10^-2
10^-3
10^-4
10^-5
2.23
36h
Plate
1
Plate
2
Survivors
Log
CFU/ml
Plate
1
Plate
2
Survivors
Log
CFU/ml
2
0
0
0
0
0
0
0
0
0
10
1.00
2
0
10
1.00
Plate
1
Plate
2
----48h
Dilution on
Plate
170
Survivors
Log
CFU/ml
-----
107
10^-1
0
10^-2
-----
10^-3
10^-4
10^-5
0
<5
<0.70
-----
Appendix IX. Inocula Raw Data, Chapter 3. Bold indicates value used for calculation.
Table IX.1 Inocula for Juice Survival Studies (Log CFU/ml represents 100 µl of inoculum used)
E. coli O157:H7 12/14/11
E. coli O157:H7 08/22/12
Dilution
on Plate
10^-6
Plate 1
Plate 2
CFU/ml
85
82
83500000
10
11
835000
10^-8
2
1
Salmonella Typhimurium 12/14/11
10^-7
Dilution
on Pate
10^-6
10^-7
10^-8
Dilution
on Plate
10^-6
10^-7
10^-8
Dilution
on Plate
10^-5
10^-6
10^-7
Dilution
on Plate
10^-6
10^-7
10^-8
Plate 1
Plate 2
CFU/ml
85
75
80000000
8
13
800000
1
0
L. monocytogenes 03/03/12
Plate 1
Plate 2
CFU/ml
208
199 245666667
33
24
2456667
2
3
S. aureus 03/23/12
Plate 1
Plate 2
CFU/ml
267
232
37733333
36
54
377333
7
4
S. aureus 09/18/12
Plate 1
Plate 2
CFU/ml
116
6
1
136
15
2
126000000
1260000
Log
CFU/ml
Plate 1
Plate 2
CFU/ml
Log
CFU/ml
124
117 240250000
5.92
36
36
2402500
4
2
Salmonella Typhimurium 08/14/12
Log
CFU/ml
5.90
Log
CFU/ml
6.39
Log
CFU/ml
5.58
Log
CFU/ml
6.10
Plate 1
62
14
3
Plate 1
272
33
8
Plate 2
CFU/ml
6.38
Log CFU/ml
57 59500000
7
595000
5.77
2
L. monocytogenes 09/05/12
Plate 2
CFU/ml
295 345000000
36
3450000
6
S. aureus 04/11/12
Log
CFU/ml
6.54
Plate 1
Plate 2
CFU/ml
Log CFU/ml
268
40
6
263
58
4
49000000
490000
5.69
108
Table IX.2 Inocula for Wine Survival Studies (Log CFU/ml Represents 100 µl of inoculum used)
E. coli O157:H7 12/09/11
E. coli O157:H7 07/30/12
Dilution
on Plate
10^-6
10^-7
10^-8
Dilution
on Plate
10^-6
10^-7
10^-8
Dilution
on Plate
10^-6
10^-7
10^-8
Dilution
on Plate
10^-6
10^-7
10^-8
Dilution
on Plate
10^-6
10^-7
10^-8
Dilution
on Plate
10^-6
10^-7
10^-8
Plate
1
Plate
2
CFU/ml
Log
CFU/ml
109
98 103500000
18
12
1035000
6.01
1
0
Salmonella Typhimurium 12/07/11
Plate
1
107
17
3
Plate
1
165
22
0
Plate
1
TNTC
139
1
Plate
1
Plate
2
CFU/ml
Log
CFU/ml
Plate
1
Plate
2
CFU/ml
Log
CFU/ml
102
136 166000000
26
20
1660000
6.22
2
3
Salmonella Typhimurium 07/22/12
Plate
1
Plate
2
CFU/ml
Log
CFU/ml
104 105500000
TNTC TNTC
245000000
19
1055000
6.02
22
27
2450000
6.39
3
0
2
L. monocytogenes 02/08/12
L. monocytogenes 02/16/12
Plate
2
Log
CFU/ml
Plate
1
Plate
2
CFU/ml
158 161500000
21
1615000
6.21
0
L. monocytogenes 08/01/12
155
22
1
159
15
1
157000000
1570000
Plate
2
CFU/ml
CFU/ml
TNTC 1465000000
154
14650000
1
S. aureus 02/14/12
Plate
2
CFU/ml
27
18
22500000
7
0
225000
0
0
S. aureus 07/22/12
Plate
1
Plate
2
CFU/ml
96
4
1
59
11
0
77500000
775000
Log
CFU/ml
6.20
Log
CFU/ml
7.17
S. aureus 02/23/12
Log
CFU/ml
Plate
1
Plate
2
CFU/ml
5.35
28
3
1
12
3
1
20000000
200000
Log
CFU/ml
5.89
Log
CFU/ml
5.30